Safe and effective biofilm inhibitory compounds and health related uses thereof

ABSTRACT

The present invention provides compounds and materials that reduce the accumulation of microorganisms a surface, by interfering with the attachment of the organisms to the surface. The compounds and materials are thus useful in preventing the formation of biofilms on surfaces in health-related environments. By preventing the formation of biofilms, the compounds formulated according to the present invention can be used in the fabrication of grafts, implants, medical devices in order to prevent infection thereof. The compounds formulated according to the present invention display an additional anticoagulant property, permitting their use in settings where decrease in blood coagulability is desirable.

BACKGROUND OF THE INVENTION

Infection is a frequent complication of many invasive surgical,therapeutic and diagnostic procedures. For procedures involvingimplantable medical devices, avoiding infection can be particularlyproblematic because can develop into biofilms, which protect themicrobes from clearing by the subject's immune system. As theseinfections are difficult to treat with antibiotics, removal of thedevice is often necessitated, which is traumatic to the patient andincreases the medical cost.

Any material left embedded in the body provides a surface foraccumulation of infectious microorganisms, particularly bacteria andoccasionally fungi. This is understood to take place through theformation of biofilms. A biofilm is a type of fouling that occurs whenmicroorganisms attach to surfaces and secrete a hydrated polymericmatrix that surrounds them. Microorganisms existing in a biofilm, termedsessile, grow in a protected environment that insulates them from attackfrom antimicrobial agents. These sessile communities can give rise tononsessile individuals, termed planktonic, which rapidly multiply anddisperse. These planktonic organisms are responsible for invasive anddisseminated infections. They are the targets of antimicrobial therapy.Conventional treatments fail to eradicate the sessile communities rootedin the biofilm. Biofilms are understood to be a frequently occurringreservoir for infectious agents. The biology of biofilms is described inmore detail in “Bacterial biofilms: a common cause of persistentinfection,” J. Costerson, P. Stewart, E. Greenberg, Science 284:1318-1322 (1999), incorporated herein by reference.

Biofilms develop preferentially on inert surfaces or on non-livingtissue, and occur commonly on medical devices and devascularized or deadtissues. Biofilms have been identified on sequestra of dead bone and onbone grafts, from which they can incite an invasive infection calledosteomyelitis that can kill even more bone. Biofilms have been alsoidentified on living, hypovascular tissues such as native heart valves,where they are responsible for the devastating infection calledendocarditis where the microorganism not only can colonize distantlocations by seeding throughout the bloodstream, but also can destroythe heart valve itself. Infections involving implanted medical devicesgenerally involve biofilms, where a sessile community provides areservoir for an invasive infection. The presence of microorganisms in abiofilm on a medical device represents contamination of that foreignbody. The elicitation by the biofilm of clinically perceptible hostresponses constitutes an infection.

The development of an infection from an area of contamination isconsistent with the natural history of biofilm growth and development.Biofilms grow slowly, in one or more locations, colonized by one or aplurality of microorganisms. The pattern of biofilm development involvesinitial attachment of a microorganism to a solid surface, the formationof microcolonies attached to the surface, and finally thedifferentiation of the microcolonies into exopolysaccharide-encasedmature biofilms. Planktonic cells are released from biofilms in anatural pattern of programmed detachment, so that the biofilm serves asa nidus for multiple, recurrent acute invasive infections. Antibioticstypically treat the infection caused by the planktonic organisms, butfail to kill those sessile organisms protected in the biofilm.

Sessile microorganisms also give rise to localized symptoms, releasingantigens and stimulating antibody production that activates the immunesystem to attack the biofilm and the area surrounding it. Antibodies andhost immune defenses are ineffective in killing the organisms in thebiofilm, even though these organisms have elicited the antibody andrelated immune response. The cytotoxic products of the host'simmunologically activated cells can be directed towards the host's owntissues. This phenomenon is seen in the mouth, where the host's responseto the dental biofilm can inflame tissues surrounding the teeth and giverise to periodontitis. This phenomenon can also give rise to localinflammation around implanted medical devices and bone resorption withloosening of orthopedic and dental implants.

While host defenses may hold invasive infections in check by controllingthe proliferation of planktonic organisms, this favorable equilibriumpresupposes an intact immune system. Many patients in a hospital settinghave compromised immune systems, rendering them more vulnerable toinvasive infections once a biofilm community has become established.Patients requiring implantable medical devices may likewise havecompromised immune systems, whether on a short-term or long-term basis.A poorly functioning immune system puts the host at greater risk forinitial formation of a contaminated biofilm around a medical device andfor the invasion of planktonic organisms into the surrounding tissuesand the system. Once the planktonic organisms mount a full-scaleinfection, the immunocompromised host will be less likely to contain andcontrol it, with potentially lethal results.

Protected from antibiotic treatment and host defenses, themicroorganisms in a biofilm typically cause recurrent infections andlow-grade local symptoms. The biofilm, once established, can only beeradicated surgically. When a foreign object becomes contaminated withmicroorganisms, the only way to eliminate local and systemic infectionmay be to remove the contaminated foreign article. If the material beingremoved is essential for health, a similar article may need to bereplaced in the same location; the replacement article will beespecially prone to infection because of the residual microorganisms inthe area.

Since the difficulties associated with eliminating biofilm-basedinfections are well-recognized, a number of technologies have developedto treat surfaces or fluids bathing surfaces to prevent or impairbiofilm formation. Biofilms adversely affect medical systems and othersystems essential to public health such as water supplies and foodproduction facilities. A number of technologies have been proposed thattreat surfaces with organic or inorganic materials to interfere withbiofilm development. For example, various methods have been employed tocoat the surfaces of medical devices with antibiotics (See e.g. U.S.Pat. Nos. 4,107,121, 4,442,133, 4,895,566, 4,917,686, 5,013,306,4,952,419, 5,853,745 and 5,902,283) and other bacteriostatic compounds(See e.g U.S. Pat. Nos. 4,605,564, 4,886,505, 5,019,096, 5,295,979,5,328,954, 5,681,575, 5,753,251, 5,770,255, and 5,877,243). Despitethese technologies, contamination of medical devices and invasiveinfection therefrom continues to be a problem.

Infectious organisms are ubiquitous in the medical environment, despitevigorous efforts to maintain antisepsis. The presence of these organismscan result in infection of hospitalized patients and medical personnel.These infections, termed nosocomial, often involve organisms morevirulent and more unusual than those encountered outside the hospital.In addition, hospital-acquired infections are more likely to involveorganisms that have developed resistance to a number of antibiotics.Although cleansing and anti-bacterial regimens are routinely employed,infectious organisms readily colonize a variety of surfaces in themedical environment, especially those surfaces exposed to moisture orimmersed in fluid. Even barrier materials, such as gloves, aprons andshields, can spread infection to the wearer or to others in the medicalenvironment. Despite sterilization and cleansing, a variety of metallicand non-metallic materials in the medical environment can retaindangerous organisms trapped in a biofilm, thence to be passed on toother hosts.

Any agent used to impair biofilm formation in the medical environmentmust be safe to the user. Certain biocidal agents, in quantitiessufficient to interfere with biofilms, also can damage host tissues.Antibiotics introduced into local tissue areas can induce the formationof resistant organisms which can then form biofilm communities whoseplanktonic microorganisms would likewise be resistant to the particularantibiotics. Any anti-biofilm or antifouling agent must furthermore notinterfere with the salubrious characteristics of a medical device.Certain materials are selected to have a particular type of operatormanipulability, softness, water-tightness, tensile strength orcompressive durability, characteristics that cannot be altered by anagent added for anti-microbial effects.

As a further problem, it is possible that materials added to thesurfaces of implantable devices to inhibit contamination and biofilmformation may be thrombogenic. Some implantable materials are ofthemselves thrombogenic. For example, it has been shown that contactwith metal, glass, plastic or other similar surfaces can induce blood toclot. Heparin compounds, which are known to have anticoagulant effects,have therefore been applied to certain medical devices prior toimplantation. However, there are few known products in the medicalarsenal whose antimicrobial effects are combined with antithrombogeniceffects. This combination would be particularly valuable to treat thosemedical devices that reside in the bloodstream, such as heart valves,artificial pumping devices (“artificial hearts” or left ventricularassist devices), vascular grafting prostheses and vascular stents. Inthese settings, clot formation can obstruct the flow of blood throughthe conduit and can furthermore break off pieces called emboli that arecarried downstream, potentially blocking circulation to distant tissuesor organs.

Biofilm formation has important public health implications. Drinkingwater systems are known to harbor biofilms, even though theseenvironments often contain disinfectants. Any system providing aninterface between a surface and a fluid has the potential for biofilmdevelopment. Water cooling towers for air conditioners are well-known topose public health risks from biofilm formation, as episodic outbreaksof infections like Legionnaires' Disease attest. Turbulent fluid flowover the surface does not provide protection: biofilms can form inconduits where flowing water or other fluids pass, with the effects ofaltering flow characteristics and passing planktonic organismsdownstream. Industrial fluid processing operations have experiencedmechanical blockages, impedance of heat transfer processes, andbiodeterioration of fluid-based industrial products, all attributable tobiofilms. Biofilms have been identified in flow conduits likehemodialysis tubing, and in water distribution conduits. Biofilms havealso been identified to cause biofouling in selected municipal waterstorage tanks, private wells and drip irrigation systems, unaffected bytreatments with up to 200 ppm chlorine.

Biofilms are a constant problem in food processing environments. Foodprocessing involves fluids, solid material and their combination. As anexample, milk processing facilities provide fluid conduits and areas offluid residence on surfaces. Cleansing milking and milk processingequipment presently utilizes interactions of mechanical, thermal andchemical processes in an air-injected clean-in-place methods.Additionally, the milk product itself is treated with pasteurization. Incheese producing, biofilms can lead to the production of calcium lactatecrystals in Cheddar cheese. Meat processing and packing facilities arein like manner susceptible to biofilm formation. Non-metallic andmetallic surfaces can be affected. Biofilms in meat processingfacilities have been detected on rubber “fingers,” plastic curtains,conveyor belt material, evisceration equipment and stainless steelsurfaces. Controlling biofilms and microorganism contamination in foodprocessing is hampered by the additional need that the agent used notaffect the taste, texture or aesthetics of the product.

SUMMARY OF THE INVENTION

In one aspect, the instant invention features medical devices andproducts comprised of a compound having the general structure 1:

wherein

X represents —OH, —O(aryl), —O(acyl), —O(sulfonyl), —CN, F, Cl, or Br;

Y represents O, S, Se, or NR;

Z represents optionally substituted alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or—(CH₂)_(m)-R₈₀;

R represents independently for each occurrence hydrogen, alkyl,heteroalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or-(CH₂)_(m)-R₈₀;

R80 represents independently for each occurrence aryl, cycloalkyl,cycloalkenyl, heterocyclyl, or polycyclyl; and m is an integer in therange 0 to 8 inclusive

By interfering with the attachment of organisms to surfaces, the instantclaimed medical devices and products have broad applicability ineffectively inhibiting a variety of organisms.

In addition, medical devices that reside in the bloodstream and arecomprised of the instant claimed compounds benefit from the combinedantimicrobial and antithrombogenic effects of the compounds.

In addition, the compounds of the invention are relatively safe, evenfor widespread use, as they naturally degrade into carbon dioxide andwater, or simple organic acids.

In addition, certain preferred compounds result in a constant orsustained release. Still other compounds have a relatively shorthalf-life following release from a surface, rendering them particularlysafe for widespread use. Yet other preferred compounds are readilysynthesized.

Particularly preferred compounds include: p-sec butylphenylchlorosulfate, p-t-butylphenyl chlorosulfate, p-t-amyphenylchlorosulfate, p-t-cumylphenyl chlorosulfate, 4-t-pentylphenylchlorosulfate, 4-octylphenyl chlorosulfate, 4-pentylphenyl acid sulfate,octyl acid sulfate, bispenyl diacid sulfate, zosteric acid, p-sulfoxycinnamic acid, p-sulfoxy ferulic acid, m,p-disulfoxy caffeic acid,benzoic sulfate, vanillic acid, gentissic acid sulfate, gallic acidsulfate and protochateuic acid, and the salts of the aforesaid acids.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a diagrammatic representation of the results of marine algaeattachment assays measuring the abundance of algal biofilm developmenton the inert coating RTV-11 compared to biofilm development on RTV-11with octyl sulfate incorporated into the coating. Relative algalabundance represents the attachment of the marine algae to the testedsurface. Error bars indicate 1 standard error of the mean (n=3) for eachtreatment. The ratio of the optical densities measured at wavelengths680 nm and 750 nm (A₆₈₀/A₇₅₀) at time O was used as a baseline referencefor all samples.

FIG. 2 is a diagrammatic representation of the results of bacterialattachment assays performed with the marine bacterium Oceanosprillum,cultured in the presence and absence of either zosteric acid or methylsulfate.

FIG. 3 is a diagrammatic representation of the results of bacterialattachment assays performed with the marine bacterium Oceanosprillum,cultured in the presence and absence of either zosteric acid or octylsulfate.

FIG. 4 is a diagrammatic representation of the results of bacterialattachment assays performed with the bacterium Alteromonas atlantica,performed in the presence and absence of either, zosteric acid, octylsulfate, or methyl sulfate.

FIG. 5 is a diagrammatic representation of the results of fungalattachment and growth assays using the fungus Aureobasidium pullulans(ashower fungus that stains grout) grown in the presence and absence ofzosteric acid, where fungal abundance represents the attachment of A.pullulans to the exposed surface.

FIG. 6 is a diagrammatic representation of the results of agglutinationof the bacterium Shewanella putrefaciens induced by the presence ofincreased amounts of zosteric acid, measured by the percent transmission(% T) of the liquid cultures at wavelength 600 mn. Agglutination isindicated by the concentration-dependent increase in % T of culturesgrown in the presence of zosteric acid. In this case, relatively highlevels of % T exhibited by the zosteric acid-exposed cultures do notreflect differences in growth, as counts of viable colony forming unitsexhibited no difference in cell density at eight hours.

FIG. 7 is a diagrammatic representation of data from prothrombinclotting time assays which displays the clotting time of erythrocytes inthe presence of high molecular weight heparin compared to the clottingtime of erythrocytes in the presence of zosteric acid.

FIG. 8 is a diagrammatic representation of data measuring the effects ofzosteric acid on the event of sea urchin egg fertilization. a) Dosedependent effect of zosteric acid on sea urchin fertilization. Percentfertilization represents a comparison of the number of eggs fertilizedin the presence of the indicated concentration of zosteric acid, to thenumber of eggs fertilized under the same conditions, in the absence ofzosteric acid. b) Relative effects of coumaric acid, heparin andzosteric acid at equal concentrations (1 mg/mL) on sea urchin eggfertilization.

FIG. 9 is a bar graph plots the fertilization of sea urchin eggs byvarious concentrations of 4 t-pentyl phenyl chlorosulfact (4-PPCS)

DETAILED DESCRIPTION OF THE INVENTION

General

In general, the present invention relates to the prevention ofaccumulation of microorganisms on any surface wherein such accumulationhas a deleterious effect on human or animal health. In particular, thepresent invention relates to the prevention of those conditionsaffecting human or animal health that involve fouling. Fouling eventsinvolve recognition between a biologic and a surface, adhesion of thebiologic to the surface, and the subsequent activity of the biologic. Asunderstood herein, the formation of a biofilm is a type of fouling.Biofilms with health effects commonly contain infectious microorganisms.

In a health-related environment, fouling can result in biofilmformation. Biofilm formation is understood to cause local contaminationof an affected area with potential for invasive local infection and forsystemic infection. Microorganisms may damage tissues in three ways: 1)they can enter or contact host cells and directly cause cell death; 2)they can release endotoxins or exotoxins that kill cells at a distance,release enzymes that degrade tissue components, or damage blood vesselsand cause ischemic necrosis; and 3) they can induce host-cellularresponses that, although directed against the invader, may causeadditional tissue damage, including suppuration, scarring andhypersensitivity reactions. An infection, whether local or systemic,represents the penetration of microorganisms into a host with theproduction of tissue damage or the elicitation of host defensemechanisms or both, leading to clinically identifiable symptoms. Commonlocal symptoms can include pain, tenderness, swelling and interferencewith function. Common systemic symptoms can include fever, malaise andhyperdynamic cardiovascular effects. Massive bloodstream invasion byinfectious agents can rapidly become fatal.

When an infection has its origins in a biofilm surrounding an object inthe body, whether a naturally occurring object or a foreign one, theinfection often cannot be controlled without removing that object. Ifthe object is naturally occurring, like devascularized or necrotictissue, it is removed surgically via a process called debridement. Ifthe object is a foreign one, such as a medical device, it is removedentirely. At times a rim of tissue must be removed along with thecontaminated object to ensure maximal removal of contaminating material.If the material being removed is essential for health, a similar articlemay need to be replaced in the same location; the replacement articlewill be especially prone to infection because of the residualmicroorganisms in the area.

Definitions

For convenience, certain terms employed in the specification, examples,and appended claims are described below.

The term “acylamino” is art-recognized and refers to a moiety that canbe represented by the general formula:

wherein R₉ is as defined above, and R′₁₁ represents a hydrogen, analkyl, an alkenyl or —(CH₂)_(m)-R₈, where m and R₈ are as defined above.

The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that comprise a double or triple bond, respectively.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group,as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as can berepresented by one of —O-alkyl, —O-alkenyl, —O-alkynyl, —O-(CH₂)_(m)-R₈,where m and R₈ are described above.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, andcycloalkyl substituted alkyl groups. In preferred embodiments, astraight chain or branched chain alkyl has 30 or fewer carbon atoms inits backbone (e.g., C₁-C₃₀ for straight chain, C₃-C₃₀ for branchedchain), and more preferably 20 or fewer. Likewise, preferred cycloalkylshave from 3-10 carbon atoms in their ring structure, and more preferablyhave 5, 6 or 7 carbons in the ring structure.

Moreover, the term “alkyl” (or “lower alkyl”) as used throughout thespecification and claims is intended to include both “unsubstitutedalkyls” and “substituted alkyls”, the latter of which refers to alkylmoieties having substituents replacing a hydrogen on one or more carbonsof the hydrocarbon backbone. Such substituents can include, for example,a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an ester, aformyl, or a ketone), a thiocarbonyl (such as a thioester, athioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphonate,a phosphinate, an amino, an amido, an amidine, an imine, a cyano, anitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, asulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or anaromatic or heteroaromatic moiety. It will be understood by thoseskilled in the art that the moieties substituted on the hydrocarbonchain can themselves be substituted, if appropriate. For instance, thesubstituents of a substituted alkyl may include substituted andunsubstituted forms of amino, azido, imino, amido, phosphoryl (includingphosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido,sulfamoyl and sulfonate), and silyl groups, as well as ethers,alkylthios, carbonyls (including ketones, aldehydes, carboxylates, andesters), —CF₃, —CN and the like. Exemplary substituted alkyls aredescribed below. Cycloalkyls can be further substituted with alkyls,alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl-substituted alkyls,—CF₃, —CN, and the like.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In preferred embodiments, the“alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl,—S-alkynyl, and —S-(CH₂)_(m)-R₈, wherein m and R₈ are defined above.Representative alkylthio groups include methylthio, ethylthio, and thelike.

The term “amido” is art recognized as an amino-substituted carbonyl andincludes a moiety that can be represented by the general formula:

wherein R₉, R₁₀ are as defined above. Preferred embodiments of the amidewill not include imides which may be unstable.

The terms “amine” and “amino” are art recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that can berepresented by the general formula:

wherein R₉, R₁₀ and R′₁₀ each independently represent a hydrogen, analkyl, an alkenyl, —(CH₂)_(m)-R₈, or R₉ and R₁₀ taken together with theN atom to which they are attached complete a heterocycle having from 4to 8 atoms in the ring structure; R₈ represents an aryl, a cycloalkyl, acycloalkenyl, a heterocycle or a polycycle; and m is zero or an integerin the range of 1 to 8. In preferred embodiments, only one of R₉ or R₁₀can be a carbonyl, e.g., R₉, R₁₀ and the nitrogen together do not forman imide. In even more preferred embodiments, R₉ and R₁₀ (and optionallyR′₁₀) each independently represent a hydrogen, an alkyl, an alkenyl, or—(CH₂)_(m)-R₈. Thus, the term “alkylamine” as used herein means an aminegroup, as defined above, having a substituted or unsubstituted alkylattached thereto, i.e., at least one of R₉ and R₁₀ is an alkyl group.

The term “anti-fouling compound” as used herein refers those chemicalformulations that impair, inhibit, prevent or retard biofouling. Ananti-fouling (“AF”) compound can be present in an acid form or as a saltthereof.

An “aprotic solvent” means a non-nucleophilic solvent having a boilingpoint range above ambient temperature, preferably from about 25° C. toabout 190° C., more preferably from about 80° C. to about 160° C., mostpreferably from about 80° C. to 150° C., at atmospheric pressure.Examples of such solvents are acetonitrile, toluene, DMF, diglyme, THFor DMSO.

The term “aryl” as used herein includes 5-, 6- and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazineand pyrimidine, and the like. Those aryl groups having heteroatoms inthe ring structure may also be referred to as “aryl heterocycles” or“heteroaromatics”. The aromatic ring can be substituted at one or morering positions with such substituents as described above, for example,halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl,amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate,carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido,ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromaticmoieties, —CF₃, —CN, or the like. The term “aryl” also includespolycyclic ring systems having two or more rings in which two or morecarbons are common to two adjoining rings (the rings are “fused”)wherein at least one of the rings is aromatic, e.g., the other rings canbe cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/orheterocyclyls.

The term “arylalkyl”, as used herein, refers to an alkyl groupsubstituted with an aryl group (e.g., an aromatic or heteroaromaticgroup).

The term “bioavailable” is meant to refer to an appropriate location,orientation or formulation of a compound for performance of thecompound's bioactivity. “Biofilm” refers to an accumulation of organismson a surface. A mature biofilm can comprise a colony of microorganismsresident upon a surface surrounded by an exopolysaccharide. “Biofilmresistant” or “antifouling” refers to inhibition or decrease in theamount of biofouling organisms that attach and/or grow.

A “biofoul or biofilm resistant coating” refers to any coating (asdefined herein) that impairs, inhibits, prevents or retards theattachment and /or growth of biofouling organisms

The term “carbocycle”, as used herein, refers to an aromatic ornon-aromatic ring in which each atom of the ring is carbon.

The term “carbonyl” is art recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R₁₁represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)-R₈ or apharmaceutically acceptable salt, R′₁₁ represents a hydrogen, an alkyl,an alkenyl or —(CH₂)_(m)-R₈, where m and R₈ are as defined above. WhereX is an oxygen and R₁₁ or R′₁₁ is not hydrogen, the formula representsan “ester”. Where X is an oxygen, and R₁₁ is as defined above, themoiety is referred to herein as a carboxyl group, and particularly whenR₁₁is a hydrogen, the formula represents a “carboxylic acid”. Where X isan oxygen, and R′₁₁ is hydrogen, the formula represents a “formate”. Ingeneral, where the oxygen atom of the above formula is replaced bysulfur, the formula represents a “thiolcarbonyl” group. Where X is asulfur and R₁₁ or R′₁₁ is not hydrogen, the formula represents a“thiolester.” Where X is a sulfur and R₁₁ is hydrogen, the formularepresents a “thiolcarboxylic acid.” Where X is a sulfur and R₁₁ ′ ishydrogen, the formula represents a “thiolformate.” On the other hand,where X is a bond, and R ₁₁ is not hydrogen, the above formularepresents a “ketone” group. Where X is a bond, and R ₁₁ is hydrogen,the above formula represents an “aldehyde” group.

“Contacting” as used herein refers to any means for providing thecompounds of the invention to a surface to be protected from biofouling.Contacting can include spraying, wetting, immersing, dipping, painting,bonding or adhering or otherwise providing a surface with a compound ofthe invention.

A “coating” refers to any temporary, semipermanent or permanent layer,covering or surface. A coating can be a gas, vapor, liquid, paste,semi-solid or solid. In addition a coating can be applied as a liquidand solidify into a hard coating. Examples of coatings include polishes,surface cleaners, caulks, adhesives, finishes, paints, waxespolymerizable compositions (including phenolic resins, siliconepolymers, chlorinated rubbers, coal tar and epoxy combinations, epoxyresin, polyamide resins, vinyl resins, elastomers, acrylate polymers,fluoropolymers, polyesters and polyurethanes, latex). Silicone resins,silicone polymers (e.g. RTV polymers) and silicone heat cured rubbersare suitable coatings for use in the invention and described for examplein the Encyclopedia of Polymer Science and Engineering (1989) 15: 204 etseq. Coatings can be ablative or dissolvable, so that the dissolutionrate of the matrix controls the rate at which AF agents are delivered tothe surface. Coatings can also be non-ablative, and rely on diffusionprinciples to deliver an AF agent to the surface. Non-ablative coatingscan be porous or non-porous. A coating containing an AF agent freelydispersed in a polymer binder is referred to as “monolithic” coating.Elasticity can be engineered into coatings to accommodate pliability,e.g. swelling or shrinkage, of the surface to be coated.

A “component” is a part of an apparatus that is structurally integratedwith that apparatus. A component may be applied to a surface of anapparatus, contained within the substance of the apparatus, retained inthe interior of the apparatus, or any other arrangement whereby thatpart is an integral element of the structure of the apparatus. As anexample, the silicone covering surrounding the mechanical part of apacemaker is a component of the pacemaker. A component may be the lumenof an apparatus where the lumen performs some function essential to theoverall function of the apparatus. The lumen of a tissue expander portis a component of the tissue expander. A component can refer to areservoir or a discrete area within the apparatus specifically adaptedfor the delivery of a fluid to a surface of the apparatus. A reservoirwithin an implantable drug delivery device is a component of thatdevice.

A “delivery system” refers to any system or apparatus or componentwhereby the disclosed antifouling compounds can be delivered to asurface upon which biofilm formation is to be inhibited. Representativedelivery systems can include encapsulation of the agent, incorporationof the agent in the substance of an article of manufacture, or insertingthe agent into the matrices or pores of a suitable object, so that theagent is able to reach the targeted surface in sufficient amount toinhibit biofilm. A delivery system can comprise a coating. A deliverysystem can comprise a mechanical object adapted for the delivery of theantifouling compound to a surface. Other mechanisms comprising deliverysystems will be apparent to those of skill in the relevant arts.

“Dressing” refer to any bandage or covering applied to a lesion orotherwise used to prevent or treat infection. Examples include wounddressings for chronic wounds (such as pressure sores, venous stasisulcers and bums) or acute wounds and dressings over percutaneous devicessuch as IVs or subclavian lines intended to decrease the risk of linesepsis due to microbial invasion. For example, the compositions of theinvention could be applied at the percutaneous puncture site, or couldbe incorporated in the adherent dressing material applied directly overthe entry site.

The phrase “effective amount” refers to an amount of the disclosedantifouling compounds that significantly reduces the number of organismsthat attach to a defined surface (cells/mm²) relative to the number thatattach to an untreated surface. Particularly preferred are amounts thatreduce the number of organisms that attach to the surface by a factor ofat least 2. Even more preferred are amounts that reduce the surfaceattachment of organisms by a factor of 4, more preferably by a factor of6. An effective amount of the disclosed antifouling compound is said toinhibit the formation of biofilms, and to inhibit the growth oforganisms on a defined surface. The term “inhibit,” as applied to theeffect of an antifouling compound on a surface includes any action thatsignificantly reduces the number of organisms that attach thereto.

The term “electron-withdrawing group” is recognized in the art, anddenotes the tendency of a substituent to attract valence electrons fromneighboring atoms, i.e., the substituent is electronegative with respectto neighboring atoms. A quantification of the level ofelectron-withdrawing capability is given by the Hammett sigma (σ)constant. This well known constant is described in many references, forinstance, J. March, Advanced Organic Chemistry, McGraw Hill BookCompany, New York, (1977 edition) pp. 251-259. The Hammett constantvalues are generally negative for electron donating groups (σ[P]=-0.66for NH₂) and positive for electron withdrawing groups (σ[P]=0.78 for anitro group), σ[P] indicating para substitution. Exemplaryelectron-withdrawing groups include nitro, ketone, aldehyde, sulfonyl,trifluoromethyl, —CN, chloride, and the like. Exemplaryelectron-donating groups include amino, methoxy, and the like.

A “graft” refers to a living tissue (e.g. skin, bone) that is introducedinto an anatomic site in a patient's body. As understood herein, theterm “graft” does not refer to those transplanted organs or tissues thatare surgically connected to a macroscopic blood supply, such as atransplanted kidney or a microsurgically vascularized fibula. Sincegrafts, as understood herein, are devascularized when they are moved,they are abnormally susceptible to infection. As grafts heal, theyacquire a blood supply and are restored to normal immune status. A graftmay be derived from the host, and is termed an autograft. A graft may bederived from a donor of the same species or from another species; suchgrafts are termed allografts and xenografts respectively.

The term “health-related environment” is understood to include all thoseenvironments where activities are carried out that are implicated in therestoration or maintenance of human health. A health-related environmentcan be a medical environment, where activities are carried out directlyor indirectly intended to restore human health. An operating room, adoctor's office, a hospital room, and a factory making medical equipmentare all examples of medical environments. Other health-relatedenvironments can include industrial or residential sites whereactivities pertaining to human health are carried out. Such activitiesinclude food processing, water purification, and sanitation.

The term “half-life” refers to the amount of time required for half of acompound to be eliminated or degraded by natural processes.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen,sulfur and phosphorous.

The terms “heterocyclyl” or “heterocyclic group” refer to 3- to10-membered ring structures, more preferably 3- to 7-membered rings,whose ring structures include one to four heteroatoms. Heterocycles canalso be polycycles. Heterocyclyl groups include, for example, thiophene,thianthrene, furan, pyran, isobenzofuran, chromene, xanthene,phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole,pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,indole, indazole, purine, quinolizine, isoquinoline, quinoline,phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline,pteridine, carbazole, carboline, phenanthridine, acridine, perimidine,phenanthroline, phenazine, phenarsazine, phenothiazine, furazan,phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine,piperazine, morpholine, lactones, lactams such as azetidinones andpyrrolidinones, sultams, sultones, and the like. The heterocyclic ringcan be substituted at one or more positions with such substituents asdescribed above, as for example, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic orheteroaromatic moiety, —CF₃, —CN, or the like.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons, more preferably from one to six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths. Preferred alkyl groups are lower alkyls. Inpreferred embodiments, a substituent designated herein as alkyl is alower alkyl.

An “implant” is any object intended for placement in a human body thatis not a living tissue. Implants include naturally derived objects thathave been processed so that their living tissues have been devitalized.As an example, bone grafts can be processed so that their living cellsare removed, but so that their shape is retained to serve as a templatefor ingrowth of bone from a host. As another example, naturallyoccurring coral can be processed to yield hydroxyapatite preparationsthat can be applied to the body for certain orthopedic and dentaltherapies. An implant can also be an article comprising artificialcomponents. The term “implant” can be applied to the entire spectrum ofmedical devices intended for placement in a human body.

The terms “infectious microorganisms” or “infectious agents” as usedherein refers to disease causing or contributing bacteria (includinggram-negative and gram-positive organisms, such as Staphylococci sps.(e.g. Staphylococcus aureus, Staphylococcus epidermis), Enterococcus sp.(E. faecalis), Pseudomonas sp. (P. aeruginosa), Escherichia sp. (E.coli), Proteus sp. (P. mirabilis) ), fungi (including Candida albicans),viruses and protists.

“Medical device” refers to a non-naturally occurring object that isinserted or implanted in a subject or applied to a surface of a subject.Medical devices can be made of a variety of biocompatible materials,including: metals, ceramics, polymers, gels and fluids not normallyfound within the human body. Examples of polymers useful in fabricatingmedical devices include such polymers as silicones, rubbers, latex,plastics, polyanhydrides, polyesters, polyorthoesters, polyamides,polyacrylonitrile, polyurethanes, polyethylene, polytetrafluoroethylene,polyethylenetetraphthalate and polyphazenes. Medical devices can also befabricated using certain naturally-occurring materials or treatednaturally-occurring materials. As an example, a heart valve can befabricated by combining a treated porcine heart valve with an affixationapparatus using artificial materials. Medical devices can include anycombination of artificial materials, combinations selected because ofthe particular characteristics of the components. For example, a hipimplant can include a combination of a metallic shaft to bear theweight, a ceramic artificial joint and a polymeric glue to affix thestructure to the surrounding bone. An implantable device is one intendedto be completely imbedded in the body without any structure left outsidethe body (e.g. heart valve). An insertable device is one that ispartially imbedded in the body but has a part intended to be external(e.g. a catheter or a drain). Medical devices can be intended forshort-term or long-term residence where they are positioned. A hipimplant is intended for several decades of use, for example. Bycontrast, a tissue expander may only be needed for a few months, and isremoved thereafter. Insertable devices tend to remain in place forshorter times than implantable devices, in part because they come intomore contact with microorganisms that can colonize them.

As used herein, the term “nitro” means —NO₂; the term “halogen”designates —F, —Cl, —Br or —I; the term “sulfhydryl” means —SH; the term“hydroxyl” means —OH; and the term “sulfonyl” means —SO₂—.

The terms ortho, meta and para apply to 1,2-, 1,3- and 1,4-disubstitutedbenzenes, respectively. For example, the names 1,2-dimethylbenzene andortho-dimethylbenzene are synonymous.

A “pharmaceutically effective amount” refers to an appropriate amount toobtain a therapeutic effect. Toxicity and therapeutic efficacy ofcompounds can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., for determining The Ld₅₀(The Dose Lethal To 50% Of The Population) And The Ed₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio LD₅₀/ED₅₀. Compounds which exhibit largetherapeutic indices are preferred. The effective amount may vary withina range depending upon the dosage form employed and the route ofadministration utilized. A dose may be formulated in animal models toachieve a circulating plasma concentration range that includes the IC₅₀(i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture.

“Pharmaceutical effective carrier” refers to a physiologicallyacceptable carriers or excipient. Thus, the compounds and theirphysiologically acceptable salts and solvates may be formulated foradministration by, for example, injection, inhalation or insufflation(either through the mouth or the nose) or oral, buccal, parenteral orrectal administration. For therapy, the compounds of the invention canbe formulated for a variety of loads of administration, includingsystemic and topical or localized administration. Techniques andformulations generally may be found in Remmington's PharmaceuticalSciences, Meade Publishing Co., Easton, Pa. For systemic administration,injection is preferred, including intramuscular, intravenous,intraperitoneal, and subcutaneous. For injection, the compounds of theinvention can be formulated in liquid solutions, preferably inphysiologically compatible buffers such as Hank's solution or Ringer'ssolution. In addition, the compounds may be formulated in solid form andredissolved or suspended immediately prior to use. Lyophilized forms arealso included. For oral administration, the pharmaceutical compositionsmay take the form of, for example, tablets or capsules prepared byconventional means with pharmaceutically acceptable excipients such asbinding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidoneor hydroxypropyl methylcellulose); fillers (e.g., lactose,microcrystalline cellulose or calcium hydrogen phosphate); lubricants(e.g., magnesium stearate, talc or silica); disintegrants (e.g., potatostarch or sodium starch glycolate); or wetting agents (e.g., sodiumlauryl sulphate). The tablets may be coated by methods well known in theart. Liquid preparations for oral administration may take the form of,for example, solutions, syrups or suspensions, or they may be presentedas a dry product for constitution with water or other suitable vehiclebefore use. Such liquid preparations may be prepared by conventionalmeans with pharmaceutically acceptable additives such as suspendingagents (e.g., sorbitol syrup, cellulose derivatives or hydrogenatededible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueousvehicles (e.g., ationd oil, oily esters, ethyl alcohol or fractionatedvegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The preparations may alsocontain buffer salts, flavoring, coloring and sweetening agents asappropriate. Preparations for oral administration may be suitablyformulated to give controlled release of the active compound. For buccaladministration the compositions may take the form of tablets or lozengesformulated in conventional manner. For administration by inhalation, thecompounds for use according to the present invention are convenientlydelivered in the form of an aerosol spray presentation from pressurizedpacks or a nebuliser, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch. The compounds may be formulated for parenteraladministration by injection, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multi-dose containers, with an addedpreservative. The compositions may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use. The compounds may also be formulated in rectal compositionssuch as suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides. In additionto the formulations described previously, the compounds may also beformulated as a depot preparation. Such long acting formulations may beadministered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt. Systemic administration can also be bytransmucosal or transdermal means. For transmucosal or transdermaladministration, penetrants appropriate to the barrier to be permeatedare used in the formulation. Such penetrants are generally known in theart, and include, for example, for transmucosal administration bilesalts and fusidic acid derivatives. in addition, detergents may be usedto facilitate permeation. Transmucosal administration may be throughnasal sprays or using suppositories. For topical administration, theoligomers of the invention are formulated into ointments, salves, gels,or creams as generally known in the art. A wash solution can be usedlocally to treat an injury or inflammation to accelerate healing.

A “phosphoryl” can in general be represented by the formula:

wherein Q₁ represented S or O, and R₄₆ represents hydrogen, a loweralkyl or an aryl. When used to substitute, e.g., an alkyl, thephosphoryl group of the phosphorylalkyl can be represented by thegeneral formula:

wherein Q₁ represented S or O, and each R₄₆ independently representshydrogen, a lower alkyl or an aryl, Q₂ represents O, S or N. When Q₁ isan S, the phosphoryl moiety is a “phosphorothioate”.

A “polar, aprotic solvent” means a polar solvent as defined above whichhas no available hydrogens to exchange with the compounds of thisinvention during reaction, for example DMF, acetonitrile, diglyme, DMSO,or THF.

A “polar solvent” means a solvent which has a dielectric constant (ε) of2.9 or greater, such as DMF, THF, ethylene glycol dimethyl ether (DME),DMSO, acetone, acetonitrile, methanol, ethanol, isopropanol, n-propanol,t-butanol or 2-methoxyethyl ether. Preferred solvents are DMF, DME, NMP,and acetonitrile.

The terms “polycyclyl” or “polycyclic group” refer to two or more rings(e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/orheterocyclyls) in which two or more carbons are common to two adjoiningrings, e.g., the rings are “fused rings”. Rings that are joined throughnon-adjacent atoms are termed “bridged” rings. Each of the rings of thepolycycle can be substituted with such substituents as described above,as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromaticmoiety, —CF₃, —CN, or the like.

The phrase “protecting group” as used herein means temporarymodifications of a potentially reactive functional group which protectit from undesired chemical transformations. Examples of such protectinggroups include esters of carboxylic acids, silyl ethers of alcohols, andacetals and ketals of aldehydes and ketones, respectively. The field ofprotecting group chemistry has been reviewed (Greene, T. W.; Wuts, P. G.M. Protective Groups in Organic Synthesis, 2nd ed.; Wiley: New York,1991).

“Release rate” or “flux” refers to the rate of delivery or diffusion ofa compound to or from a surface. The release rate may be constant orsustained over a period of time or may be variable. However, constant,controlled or sustained release rates are generally preferred. Steadystate or sustained release may be effected by use of a reservoirmembrane (i.e. a two layer coating in which one layer contains theactive agent and the other creates a membrane through which the activeagent can be released). The active agent could alternatively bemicroencapsulated within any of a variety of matrices for sustainedrelease. Preferred release rates for applications that require a shortduration of AF activity or that are frequently applied are more thanabout 100 μgcm⁻²d⁻¹. Preferred release rates for sustained release aretypically less than 100 μgcm⁻²d^(−1,) more preferably less than about75, 50, 25, 20, 15, 10 or 5 μgcm⁻²d⁻¹.

The term “soluble” refers to the ability to be loosened or dissolved.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described herein above. The permissible substituentscan be one or more and the same or different for appropriate organiccompounds. For purposes of this invention, the heteroatoms such asnitrogen may have hydrogen substituents and/or any permissiblesubstituents of organic compounds described herein which satisfy thevalences of the heteroatoms. This invention is not intended to belimited in any manner by the permissible substituents of organiccompounds.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc.

The term “sulfate” is art recognized and includes a moiety that can berepresented by the general formula:

in which R₄₁ is as defined above.

A “sulfate binding moiety” refers to a moiety that is capable of bindingor otherwise associating with a sulfate or sulfonate group.

The term “sulfonate” is art recognized and includes a moiety that can berepresented by the general formula:

in which R₄₁ is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The terms “sulfoxido” or “sulfinyl”, as used herein, refers to a moietythat can be represented by the general formula:

in which R₄₄ is selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclyl, aralkyl, or aryl.

Analogous substitutions can be made to alkenyl and alkynyl groups toproduce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls,amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls,carbonyl-substituted alkenyls or alkynyls.

The term “surface”, as used herein, refers to any surface whether in anindustrial or a medical setting, that provides an interface between anobject and a fluid, permitting at least intermittent contact between theobject and the fluid. A surface, as understood herein, further providesa plane whose mechanical structure, without further treatment, iscompatible with the adherence of microorganisms. Surfaces compatiblewith biofilm formation may be smooth or irregular. Fluids contacting thesurfaces can be stagnant or flowing, and can flow intermittently orcontinuously, with laminar or turbulent or mixed rheologies. A surfaceupon which a biofilm forms can be dry at times with sporadic fluidcontact, or can have any degree of fluid exposure including totalimmersion. Fluid contact with the surface can take place via aerosols orother means for air-borne fluid transmission.

Biofilm formation with health implications can involve those surfaces inall health-related environments, including surfaces found in medicalenvironments and those surfaces in industrial or residentialenvironments that are involved in those functions essential towell-being like nutrition, sanitation and the prevention of disease.

A surface of an article adapted for use in a medical environment can becapable of sterilization using autoclaving, biocide exposure,irradiation or gassing techniques like ethylene oxide exposure. Surfacesfound in medical environments include the inner and outer aspects ofvarious instruments and devices, whether disposable or intended forrepeated uses. Examples include the entire spectrum of articles adaptedfor medical use, including scalpels, needles, scissors and other devicesused in invasive surgical, therapeutic or diagnostic procedures;implantable medical devices, including artificial blood vessels,catheters and other devices for the removal or delivery of fluids topatients, artificial hearts, artificial kidneys, orthopedic pins, platesand implants; catheters and other tubes (including urological andbiliary tubes, endotracheal tubes, peripherably insertable centralvenous catheters, dialysis catheters, long term tunneled central venouscatheters, peripheral venous catheters, short term central venouscatheters, arterial catheters, pulmonary catheters, Swan-Ganz catheters,urinary catheters, peritoneal catheters), urinary devices (includinglong term urinary devices, tissue bonding urinary devices, artificialurinary sphincters, urinary dilators), shunts (including ventricular orarterio-venous shunts); prostheses (including breast implants, penileprostheses, vascular grafting prostheses, heart valves, artificialjoints, artificial larynxes, otological implants), vascular catheterports, wound drain tubes, hydrocephalus shunts, pacemakers andimplantable defibrillators, and the like. Other examples will be readilyapparent to practitioners in these arts.

Surfaces found in the medical environment include also the inner andouter aspects of pieces of medical equipment, medical gear worn orcarried by personnel in the health care setting. Such surfaces caninclude counter tops and fixtures in areas used for medical proceduresor for preparing medical apparatus, tubes and canisters used inrespiratory treatments, including the administration of oxygen, ofsolubilized drugs in nebulizers and of anesthetic agents. Also includedare those surfaces intended as biological barriers to infectiousorganisms in medical settings, such as gloves, aprons and faceshields.Commonly used materials for biological barriers may be latex-based ornon-latex based. Vinyl is commonly used as a material for non-latexsurgical gloves. Other such surfaces can include handles and cables formedical or dental equipment not intended to be sterile. Additionally,such surfaces can include those non-sterile external surfaces of tubesand other apparatus found in areas where blood or body fluids or otherhazardous biomaterials are commonly encountered.

Surfaces in contact with liquids are particularly prone to biofilmformation. As an example, those reservoirs and tubes used for deliveringhumidified oxygen to patients can bear biofilms inhabited by infectiousagents. Dental unit waterlines similarly can bear biofilms on theirsurfaces, providing a reservoir for continuing contamination of thesystem of flowing and aerosolized water used in dentistry.

Sprays, aerosols and nebulizers are highly effective in disseminatingbiofilm fragments to a potential host or to another environmental site.It is understood to be especially important to health to prevent biofilmformation on those surfaces from whence biofilm fragments can be carriedaway by sprays, aerosols or nebulizers contacting the surface.

Other surfaces related to health include the inner and outer aspects ofthose articles involved in water purification, water storage and waterdelivery, and those articles involved in food processing. Surfacesrelated to health can also include the inner and outer aspects of thosehousehold articles involved in providing for nutrition, sanitation ordisease prevention. Examples can include food processing equipment forhome use, materials for infant care, tampons and toilet bowls.

“Sustained release” or “controlled release refers to a relativelyconstant or prolonged release of a compound of the invention from asurface. This can be accomplished through the use of diffusionalsystems, including reservoir devices in which a core of a compound ofthe invention is surrounded by a porous membrane or layer, and alsomatrix devices in which the compound is distributed throughout an inertmatrix. Materials which may be used to form reservoirs or matricesinclude silicones, acrylates, methacrylates, vinyl compounds such aspolyvinyl chloride, olefins such as polyethylene or polypropylene,fluoropolymers such as polytetrafluorethylene, and polyesters such asterephthalates. The diffusional systems may be molded into a film orother layer material which is then placed in adherent contact with thestructure intended for underwater use. Alternatively, the compounds ofthe invention may be mixed with a resin, e.g., polyvinyl chloride andthen molded into a formed article, which integrally incorporates thecompound to form a structure having a porous matrix which allowsdiffusion of the compound, or a functional portion thereof, into thesurrounding environment. Microencapsulation techniques can also be usedto maintain a sustained focal release of a compound of the invention.Microencapsulation may also be used for providing improved stability.The encapsulated product can take the form of, for example, spheres,aggregates of core material embedded in a continuum of wall material, orcapillary designs. The core material of a microcapsule containing asulfate ester AF agent may be in the form of a liquid droplet, anemulsion, a suspension of solids, a solid particle, or a crystal. Theskilled artisan will be aware of numerous materials suitable for use asmicrocapsule coating materials, including, but not limited to, organicpolymers, hydrocolloids, lipids, fats, carbohydrates, waxes, metals, andinorganic oxides. Silicone polymers are the most preferred microcapsulecoating material for treatment of surfaces. Microencapsulationtechniques are well known in the art and are described in theEncyclopedia of Polymer Science and Engineering, Vol. 9, pp. 724 et seq.(1989) hereby incorporated by reference.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms, and dba represent methyl,ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,p-toluenesulfonyl, methanesulfonyl, and dibenzylideneacetone,respectively. A more comprehensive list of the abbreviations utilized byorganic chemists of ordinary skill in the art appears in the first issueof each volume of the Journal of Organic Chemistry; this list istypically presented in a table entitled Standard List of Abbreviations.The abbreviations contained in said list, and all abbreviations utilizedby organic chemists of ordinary skill in the art are hereby incorporatedby reference.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover. Alsofor purposes of this invention, the term “hydrocarbon” is contemplatedto include all permissible compounds having at least one hydrogen andone carbon atom. In a broad aspect, the permissible hydrocarbons includeacyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and nonaromatic organic compounds which can besubstituted or unsubstituted.

Compositions of the Invention

In certain embodiments, the compositions of the present inventioncomprise an anti-fouling compound represented by general structure 1:

wherein

X represents —OH, —O(aryl), —O(acyl), —O(sulfonyl), —CN, F, Cl, or Br;

Y represents O, S, Se, or NR;

Z represents optionally substituted alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or—(CH₂)_(m)-R₈₀;

R represents independently for each occurrence hydrogen, alkyl,heteroalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or—(CH₂)_(m)-R₈₀;

R₈₀ represents independently for each occurrence aryl, cycloalkyl,cycloalkenyl, heterocyclyl, or polycyclyl; and

m is an integer in the range 0 to 8 inclusive.

Particularly stable compounds are represented by general structure 1 andthe attendant definitions, wherein X represents —OH, F, Cl, or Br.

In other preferred embodiments, the compositions of the presentinvention comprise an anti-fouling compound represented by generalstructure 1 and the attendant definitions, wherein Y represents O.

In certain embodiments, the compositions of the present inventioncomprise an anti-fouling compound represented by general structure 1 andthe attendant definitions, wherein Z represents optionally substitutedalkyl, aryl, or —(CH₂)_(m)-R₈₀.

In certain embodiments, the compositions of the present inventioncomprise an anti-fouling compound represented by general structure 1 andthe attendant definitions, wherein Z represents optionally substitutedalkylphenyl, heteroalkylphenyl, arylphenyl, or heteroarylphenyl.

In certain embodiments, the compositions of the present inventioncomprise an anti-fouling compound represented by general structure 1 andthe attendant definitions, wherein Z represents4-(2-methylpropyl)phenyl, 4-(1, 1 -dimethylethyl)phenyl, 4-(1, 1-dimethylpropyl)phenyl, 4-pentylphenyl, 4-(1-methyl- 1-phenylethyl)phenyl, or 4-(1 -methylheptyl)phenyl.

In certain embodiments, the compositions of the present inventioncomprise an anti-fouling compound represented by general structure 1 andthe attendant definitions, wherein R represents H or alkyl.

In certain embodiments, the compositions of the present inventioncomprise an anti-fouling compound represented by general structure 1 andthe attendant definitions, wherein X represents —OH, F, Cl, or Br; and Yrepresents O.

In certain embodiments, the compositions of the present inventioncomprise an anti-fouling compound represented by general structure 1 andthe attendant definitions, wherein X represents —OH or Cl; and Yrepresents O.

In certain embodiments, the compositions of the present inventioncomprise an anti-fouling compound represented by general structure 1 andthe attendant definitions, wherein X represents —OH, F, Cl, or Br; and Zrepresents optionally substituted alkyl, aryl, or —(CH₂)_(m)-R₈₀.

In certain embodiments, the compositions of the present inventioncomprise an anti-fouling compound represented by general structure 1 andthe attendant definitions, wherein X represents —OH or Cl; and Zrepresents optionally substituted alkyl, aryl, or —(CH₂)_(m)—R₈₀.

In certain embodiments, the compositions of the present inventioncomprise an anti-fouling compound represented by general structure 1 andthe attendant definitions, wherein X represents —OH, F, Cl, or Br; and Zrepresents optionally substituted alkylphenyl, heteroalkylphenyl,arylphenyl, or heteroarylphenyl.

In certain embodiments, the compositions of the present inventioncomprise an anti-fouling compound represented by general structure 1 andthe attendant definitions, wherein X represents —OH or Cl; and Zrepresents optionally substituted alkylphenyl, heteroalkylphenyl,arylphenyl, or heteroarylphenyl.

In certain embodiments, the compositions of the present inventioncomprise an anti-fouling compound represented by general structure 1 andthe attendant definitions, wherein X represents —OH, F, Cl, or Br; and Zrepresents 4-(2-methylpropyl)phenyl, 4-(1,1-dimethylethyl)phenyl, 4-(1,1-dimethylpropyl)phenyl, 4-pentylphenyl,4-(1-methyl-1-phenylethyl)phenyl, or 4-(1-methylheptyl)phenyl.

In certain embodiments, the compositions of the present inventioncomprise an anti-fouling compound represented by general structure 1 andthe attendant definitions, wherein X represents —OH or Cl; and Zrepresents 4-(2-methylpropyl)phenyl, 4-(1,1-dimethylethyl)phenyl, 4-(1,1-dimethylpropyl)phenyl, 4-pentylphenyl,4-(1-methyl-1-phenylethyl)phenyl, or 4-(1-methylheptyl)phenyl.

In certain embodiments, the compositions of the present inventioncomprise an anti-fouling compound represented by general structure 1 andthe attendant definitions, wherein Y represents O; and Z representsoptionally substituted alkyl, aryl, or —(CH₂)_(m)-R₈₀.

In certain embodiments, the compositions of the present inventioncomprise an anti-fouling compound represented by general structure 1 andthe attendant definitions, wherein Y represents O; and Z representsoptionally substituted alkylphenyl, heteroalkylphenyl, arylphenyl, orheteroarylphenyl.

In certain embodiments, the compositions of the present inventioncomprise an anti-fouling compound represented by general structure 1 andthe attendant definitions, wherein Y represents O; and Z represents4-(2-methylpropyl)phenyl, 4-(1,1 -dimethylethyl)phenyl,4-(1,1-dimethylpropyl)phenyl, 4-pentylphenyl,4-(1-methyl-1-phenylethyl)phenyl, or 4-(1-methylheptyl)phenyl.

In certain embodiments, the compositions of the present inventioncomprise an anti-fouling compound represented by general structure 1 andthe attendant definitions, wherein X represents —OH, F, Cl, or Br; Yrepresents O; and Z represents optionally substituted alkyl, aryl, or—(CH₂)_(m)-R₈₀.

In certain embodiments, the compositions of the present inventioncomprise an anti-fouling compound represented by general structure 1 andthe attendant definitions, wherein X represents —OH or Cl; Y representsO; and Z represents optionally substituted alkyl, aryl, or—(CH₂)_(m)-R₈₀.

In certain embodiments, the compositions of the present inventioncomprise an anti-fouling compound represented by general structure 1 andthe attendant definitions, wherein X represents —OH, F, Cl, or Br; Yrepresents O; and Z represents optionally substituted alkylphenyl,heteroalkylphenyl, arylphenyl, or heteroarylphenyl.

In certain embodiments, the compositions of the present inventioncomprise an anti-fouling compound represented by general structure 1 andthe attendant definitions, wherein X represents —OH or Cl; Y representsO; and Z represents optionally substituted alkylphenyl,heteroalkylphenyl, arylphenyl, or heteroarylphenyl.

In certain embodiments, the compositions of the present inventioncomprise an anti-fouling-compound represented by general structure 1 andthe attendant definitions, wherein X represents —OH, F, Cl, or Br; Yrepresents O; and Z represents 4-(2-methylpropyl)phenyl,4-(1,1-dimethylethyl)phenyl, 4-(1,1 -dimethylpropyl)phenyl,4-pentylphenyl, 4-(1-methyl-1-phenylethyl)phenyl, or4-(1-methylheptyl)phenyl.

In certain embodiments, the compositions of the present inventioncomprise an anti-fouling compound represented by general structure 1 andthe attendant definitions, wherein X represents —OH or Cl; Y representsO; and Z represents 4-(2-methylpropyl)phenyl,4-(1,1-dimethylethyl)phenyl, 4-(1,1-dimethylpropyl)phenyl,4-pentylphenyl, 4-(1-methyl-1-phenylethyl)phenyl, or4-(1-methylheptyl)phenyl.

One of skill in the art will recognize that the composition of theinvention can be varied as required to optimize the overall chemicalproperties of the particular compound for specific uses, while retainingthe AF activity. For example, the length of an alkyl chain can beextended or shortened to control the rate of dissolution of the compoundfrom a structure or a coating. Alternatively, additional functionalgroups can be added to the alkyl chain to further vary the chemicalnature of the molecule.

Specific Utilities

Anticoagulant Activity

As shown in the following examples, compounds of the invention have beenfound to possess certain anti-coagulation activities, such as possessedby the sulfated mucopolysaccharide heparin. Heparin is believed toinhibit the clotting cascade by binding to, and thereby activating,antithrombin III, a plasma protein that inactivates thrombin by formingan irreversible complex with it. This complex is similar to theacyl-enzyme complex formed between trypsin and pancreatic trypsininhibitor. Heparin is produced by mast cells near the walls of bloodvessels and acts as an anticoagulant by increasing the rate of formationof the irreversible complex between thrombin and antithrombin III.Antithrombin III also inhibits other proteolytic components of theclotting cascade, such as Factors IX_(a), X_(a), and XI_(a).

The activity for the low molecular weight zosteric acid molecule hasbeen observed to be considerably less than that observed for heparin.Heparin was effective at preventing clot formation at concentrationswell below 0.1 mg/ml, while zosteric acid was effective only atconcentrations exceeding 10 mg/ml. However, the effectiveness ofheparin-like anticoagulants is strongly linked to size, with highmolecular weight molecules being more effective. Therefore certain ofthe higher molecular weight compounds of the invention should prove moreeffective.

Heparin has been used extensively for prophylaxis and treatment of deepvein thrombosis. However, heparin has several limitations. For example,heparin, a relatively large molecule, has shown limited efficacy ininhibiting thrombin activity incorporated into a fibrin clot. Inaddition, heparin has a short intravenous half-life. Therefore, incertain applications, the use of compounds of the invention can beadvantageous in performing certain anti-clotting prophylaxis ortherapies. The compounds of the invention can also be formulated ascovalent derivatized for the treatment and prevention of clottingconditions. For example, a covalent antithrombin-heparin complex hasproven to be more effective in reducing clot weight in vivo thanthrombin and heparin combination therapy. Accordingly, the inventionprovides methods and reagents for creating derivatized components of thefibrin clotting cascade, including antithrombin.

The compounds of the invention can potentially be orally delivered,whereas heparin must be delivered intravenously. In addition, theheparin-like property of the compounds of the invention render themparticularly suitable for incorporating into medical materials where ananti-coagulant effect is desired.

Contraceptive Activity

As shown in the following examples, compounds of the invention have beenfound to inhibit fertilization. As a result, the compounds of theinvention can be used as contraceptives. Contraceptives comprised of theinstant claimed compounds can be formulated as either routinelyreapplied coatings, described above, foams, jellies and suppositories,or alternatively as semi-permanent coatings to be used on prophylacticarticles of manufacture, (e.g., condoms or diaphragms). Alternatively,sulfate esters can be formulated as incorporations into such articlesduring manufacture.

Biofilm Inhibitory Activities

By interfering with the attachment of organisms to surfaces, the instantclaimed compounds have broad applicability in effectively inhibiting thedevelopment of biofilms on medical and health related surfaces.

For example, wounds where skin grafts or skin substitutes such ascultured epidermal cells have been applied are particularly vulnerableto invasion by ambient infectious organisms. These wound covers have nointrinsic blood supply, so are unable to fight off infection. Whencertain organisms invade a skin graft site, they destroy the skin graftentirely. Infectious organisms can similarly invade a wound where partof the skin has been removed, either through trauma or as part of atreatment; local infection of such a partial-thickness wound can killthe skin entirely and prevent it from healing or regenerating. In thiscontext, a wound able to heal itself has been converted to one that willheal extremely slowly if at all, likely requiring a skin graft forclosure. These wounds are particularly adapted for the application of anAF agent that will reduce the tendency for infectious organisms toaccumulate without impacting the general healing process. Formulation ofsuch a preparation is consistent with the skill of ordinary practitionerin these arts.

Vehicles comprised of claimed compounds of the invention adapted forskin or tissue application can include materials for temporaryapplication, or those materials that are more durable, forming films orsemi-permanent coatings like paints. Creams and ointments can be formedthat are occlusive or that are semi-permeable.

Compounds of the invention can be formulated as a solution suitable forapplying to skin surfaces that will form a durable film that can remainin place over a sustained period of time. Such a solution could beapplied to the hands of medical personnel underneath surgical gloves toreduce the contamination hazards from glove tears. Such a solution couldalso be applied to exposed skin surfaces, for example the uncoveredface, of medical personnel in settings where contaminated splashes arelikely. This embodiment would be useful for personnel like emergencymedical technicians and emergency room doctors and nurses. A vehicle forapplying compounds of the invention like a lotion or a cream wouldprotect exposed surfaces of those personnel, and reduce the risk and thefear of possible infection from topical exposures to contaminatedfluids.

For household use, compounds of the invention can be incorporated intoointments to protect injured areas and to protect intact skin fromprolonged microbial exposure. As an example, a topical compound of theinvention can inhibit the development of fungal infections likeathlete's foot; the agent can be dispensed as a cream, an ointment, apowder or a spray. Other preparations can be used in moist areas toinhibit local yeast infections. Preparations adapted for intravaginaluse can provide prophylaxis against Candida vaginitis in patients takingbroad-spectrum antibiotics, where alteration of the normal balance offlora can result in Candida overgrowth and subsequent symptomatology.Applying a compound of the invention to the materials used forfabricating menstrual tampons may inhibit the formation of those Staph.species responsible for toxic shock syndrome.

The non-toxic nature of the compounds of the invention makes themsuitable for direct application to living tissues. A liquid preparationmay be applied to autogenous skin or bone grafts to protect thesetemporarily avascular tissues from microbial colonization and biofilmformation. The vehicle bearing the compound need only remain present andbiologically active for a short period of time, because the graftedtissues will soon begin to acquire a blood supply, therewith acquiringmore normal local immune responsiveness.

Any object placed in the body from outside it is susceptible tobiological contamination with microorganisms and subsequent biofilmformation. Therefore, compounds according to the present invention canprevent such objects from becoming contaminated with microorganisms inthe first place. Further, these technologies can be used to producenatural and synthetic materials resistant to contamination that areespecially suited for replacing those objects that have alreadysustained infection, or that are intended for being placed in thoseanatomic sites where infections can be particularly devastating.

Naturally derived processed materials commonly are positioned in thebody in order to provide a structure for ingrowth of the patient's owntissues. Examples include demineralized bone materials andhydroxyapatite. These materials are destined to be infiltrated orreplaced entirely by the patient's tissue, during which time theexogenous material retains the desired shape or structural support inthe affected area. These materials themselves are non-living andavascular. Colonization of these materials with microorganisms andbiofilm formation can require their removal. If the material is removed,the shape or the structure that it is maintaining is destroyed and theprogress made by tissue ingrowth is in vain. Application of compounds ofthe invention to these materials can enhance their resistance to biofilmformation and its consequences.

Certain naturally derived processed materials will be determined byartisans in these fields to especially suitable for the application orincorporation of compounds of the invention. A material can be contactedwith the claimed compounds in a variety of ways including immersion andcoating. In forms where the material has interstices, an AF compound canreside therein as a liquid or as a gel. Fibrillar preparations canpermit the fibers to be coated with the compound. Solid articles such asreconstructive blocks of hydroxyapatite can be painted with a coating ofthe compound for additional protection. These temporary means ofapplication are appropriate for these materials because they only residein the body temporarily, to be resorbed or replaced.

Implantable medical devices, using artificial materials alone or incombination with naturally-derived materials, can be treated withcompounds either by surface coating or by incorporation. Metals may besuitably treated with surface coats while retaining their biologicalproperties. In certain embodiments of the present invention, metals maybe treated with paints or with adherent layers of polymers or ceramicsthat incorporate the compounds of the invention. Certain embodimentstreated in this manner may be suitable for orthopedic applications, forexample, pins, screws, plates or parts of artificial joints. Methods forsurface treatment of metals for biological use are well-known in therelevant arts. Other materials besides metals can be treated withsurface coats of compounds according to the present invention as themedical application requires.

Implantable devices may comprise materials suitable for theincorporation of the instant claimed compounds. Embodiments whosecomponents incorporate compounds of the invention can include polymers,ceramics and other substances. Materials fabricated from artificialmaterials can also be destined for resorption when they are placed inthe body. Such materials can be called bioabsorbable. As an example,polyglycolic acid polymers can be used to fabricate sutures andorthopedic devices. Those of ordinary skill in these arts will befamiliar with techniques for incorporating agents into the polymers usedto shape formed articles for medical applications. AF agents can also beincorporated into glues, cements or adhesives, or in other materialsused to fix structures within the body or to adhere implants to a bodystructure. Examples include polymethylmethacrylate and its relatedcompounds, used for the affixation of orthopedic and dental prostheseswithin the body. The presence of the compounds of the instant inventioncan decrease biofilm formation in those structures in contact with theglue, cement, or adhesive. Alternatively, a compound of the inventioncan coat or can permeate the formed article. In these compositions, theformed article allows diffusion of the compound, or functional portionthereof, into the surrounding environment, thereby preventing fouling ofthe appliance itself. Microcapsules bearing compounds can also beimbedded in the material. Materials incorporating compounds areadaptable to the manufacture of a wide range of medical devices, some ofwhich are disclosed below. Other examples will be readily apparent tothose practitioners of ordinary skill in the art.

In one embodiment, compounds of the invention can be applied to orincorporated in certain medical devices that are intended to be left inposition permanently to replace or restore vital functions. As oneexample, ventriculoatrial or ventriculoperitoneal shunts are devised toprevent cerebrospinal fluid from collecting in the brain of patientswhose normal drainage channels are impaired. As long as the shuntfunctions, fluid is prevented from accumulating in the brain and normalbrain function can continue. If the shunt ceases to function, fluidaccumulates and compresses the brain, with potentially life-threateningeffect. If the shunt becomes infected, it causes an infection to enterthe central portions of the brain, another life-threateningcomplication. These shunts commonly include a silicone elastomer oranother polymer as part of their fabrication. Silicones are understoodto be especially suited for combination with compounds according to thepresent invention.

Another shunt that has life-saving import is a dialysis shunt, a pieceof polymeric tubing connecting an artery and a vein in the forearm toprovide the kidney failure patient a means by which the dialysisequipment can cleanse the bloodstream. Even though this is a high-flowconduit, it is susceptible to the formation of biofilms and subsequentinfection. If a shunt becomes infected, it requires removal andreplacement. Since dialysis may be a lifelong process, and since thereare a limited number of sites where shunts can be applied, it isdesirable to avoid having to remove one through infectiouscomplications. Imbedding or otherwise contacting the compounds of theinvention with the shunt material can have this desired effect.

Heart valves comprising artificial material are understood to bevulnerable to the dangerous complication of prosthetic valveendocarditis. Once established, it carries a mortality rate as high as70%. Biofilms are integrally involved in the development of thiscondition. At present, the only treatment for established contaminationis high-dose antibiotic therapy and surgical removal of the device. Thecontaminated valve must be immediately replaced, since the heart cannotfunction without it. Because the new valve is being inserted in arecently contaminated area, it is common for prosthetic valveendocarditis to affect the replacement valve as well. Artificial heartvalves comprised of the compounds of the invention may reduce theincidence of primary and recurrent prosthetic valve endocarditis.Compounds of the invention can be applied to the synthetic portions orthe naturally-derived portions of heart valves.

Pacemakers and artificial implantable defibrillators commonly comprisemetallic parts in combination with other synthetic materials. Thesedevices, which may be coated with a polymeric substance such as siliconeare typically implanted in subcutaneous or intramuscular locations withwires or other electrical devices extending intrathoracically orintravascularly. If the device becomes colonized with microorganisms andinfected, it must be removed. A new device can be replaced in adifferent location, although there are a finite number of appropriateimplantation sites on the body. Devices comprising the compounds of theinvention may inhibit contamination and infection, or substantiallyreduce the risk thereof.

Devices implanted into the body either temporarily or permanently topump pharmacological agents into the body can comprise metallic parts incombination with other synthetic materials. Such devices, termedinfusion pumps, can be entirely implanted or can be partially implanted.The device may be partially or entirely covered with a polymericsubstance, and may comprise other polymers used as conduits or tubes.Incorporating AF agents according to the present invention into thecoating materials imposed upon these devices or into the materials usedfor the devices themselves, their conduits or their tubing may inhibittheir contamination and infection.

Equally lifesaving are the various vascular grafting prostheses andstents intended to bypass blocked arteries or substitute for damagedarteries. Vascular grafting prostheses, made of Teflon, dacron,Gore-tex®, expanded polytetrafluoroethylene (e-PTFE), and relatedmaterials, are available for use on any major blood vessel in the body.Commonly, for example, vascular grafting prostheses are used to bypassvessels in the leg and are used to substitute for a damaged aorta. Theyare put in place by being sewn into the end or the side of a normalblood vessel upstream and downstream of the area to be bypassed orreplaced, so that blood flows from a normal area into the vasculargrafting prosthesis to be delivered to other normal blood vessels.Stents comprising metallic frames covered with vascular graftingprosthesis fabric are also available for endovascular application, torepair damaged blood vessels.

When a vascular grafting prosthesis becomes infected, it can spreadinfection through the entire bloodstream. Furthermore, the infection canweaken the attachment of the vascular grafting prosthesis to the normalblood vessel upstream or downstream, so that blood can leak out of it.If the attachment ruptures, there can be life-threatening hemorrhage.When a vascular grafting prosthesis becomes infected, it needs to beremoved. It is especially dangerous to put another vascular graftingprosthesis in the same spot because of the risk of another infection,but there are often few other options. Vascular grafting prosthesescomprising compounds of the invention can resist infections, therebyavoiding these devastating complications.

Vascular grafting prostheses of small caliber are particularly prone toclotting. A vascular grafting prosthesis comprising a compound of theinvention may not only prevent biofilm formation, but also inhibitclotting as described above, allowing a smaller diameter vasculargrafting prosthesis to be more reliable. A common site for clotting isthe junction point between the vascular grafting prosthesis and thenormal vessel, called the anastomosis. Even if an artificial vasculargrafting prosthesis is not used, anywhere that two vessels are joined oranywhere there is a suture line that penetrates a natural blood vessel,there is a potential for clotting to take place. A clot in a vessel canocclude the vessel entirely or only partially; in the latter case, bloodclots can be swept downstream, damaging local tissues. Using suturecomprised of the compounds of the invention may inhibit clotting atthese various suture lines. The smaller the vessel, the more likely thata clot forming within it will lead to a total occlusion. This can havedisastrous results: if the main vessel feeding a tissue or an organbecomes totally occluded, that structure loses its blood supply and candie. Microsurgery provides dramatic examples of the damage that canoccur with anastomotic clotting. In microsurgery, typically only asingle tiny vessel is feeding an entire tissue structure like a fingeror a muscle. If the vessel clots off, the tissue structure can quicklydie. Microsurgery typically involves vessels only one to fourmillimeters in diameter. It is understood that the sutures penetratingthe vessel at the anastomosis are likely sites for clots to form.Microsurgical sutures comprising a compound of the invention wouldresult in localized administration of an anticoagulant at the site mostlikely to be damaged by clotting.

Suture material used to anchor vascular grafting prostheses to normalblood vessels or to sew vessels or other structures together can alsoharbor infections. Sutures used for these purposes are commonly made ofprolene, nylon or other monofilamentous nonabsorbable materials. Aninfection that begins at a suture line can extend to involve thevascular grafting prosthesis. Suture materials comprising a compound ofthe invention would have increased resistance to infection.

A suture comprising a compound of the invention would be useful in otherareas besides the vasculature. Wound infections at surgical incisionsmay arise from microorganisms that lodge in suture materials placed atvarious levels to close the incision. General surgery uses bothnonabsorbable and absorbable sutures. Materials for nonabsorbablesutures include prolene and nylon. Absorbable sutures include materialslike treated catgut and polyglycolic acid. Absorbable sutures retaintensile strength for periods of time from days to months and aregradually resorbed by the body. Fabricating an absorbable or anonabsorbable suture comprising a compound of the invention and whichretains the handling and tensile characteristics of the material iswithin the skill of artisans in the field.

A general principle of surgery is that when a foreign object becomesinfected, it most likely needs to be removed so that the infection canbe controlled. So, for example, when sutures become infected, they mayneed to be surgically removed to allow the infection to be controlled.Any area where surgery is performed is susceptible to a wound infection.Wound infections can penetrate to deeper levels of the tissues toinvolve foreign material that has been used as part of the operation. Asan example, hernias are commonly repaired by suturing a plasticscreening material called mesh in the defect. A wound infection thatextends to the area where the mesh has been placed can involve the meshitself, requiring that the mesh be removed. Surgical meshes comprising acompound of the invention can have increased resistance to infection.Surgical meshes are made of substances like Gore-tex®, teflon, nylon andMarlex®. Surgical meshes are used to close deep wounds or to reinforcethe enclosure of body cavities. Removing an infected mesh can leave anirreparable defect, with life-threatening consequences. Avoidinginfection of these materials is of paramount importance in surgery.Materials used for meshes and related materials can be formulated toinclude the claimed compounds of the instant invention.

Materials similar to vascular grafting prostheses and surgical meshesare used in other sites in the body. Medical devices used in theselocations similarly can benefit from the compounds of the invention;when these devices are located in the bloodstream, theseagents'anticoagulant effects provide further benefit. Examples includehepatic shunts, vena caval filters and atrial septal defect patches,although other examples will be apparent to practitioners in these arts.

Certain implantable devices intended to restore structural stability tobody parts can be advantageously treated with the instant claimedcompounds. A few examples follow, and others will be readily identifiedby ordinary skilled artisans. Implantable devices, used to replace bonesor joints or teeth, act as prostheses or substitutes for the normalstructure present at that anatomic site. Metallics and ceramics arecommonly used for orthopedic and dental prostheses. Implants may beanchored in place with cements like polymethylmethacrylate. Prostheticjoint surfaces can be fabricated from polymers such as silicones orteflon. Entire prosthetic joints for fingers, toes or wrists can be madefrom polymers.

Medical prostheses comprising compounds of the invention would beexpected to have reduced contamination and subsequent local infection,thereby obviating or reducing the need to remove the implant with theattendant destruction of local tissues. Destruction of local tissues,especially bones and ligaments, can make the tissue bed less hospitablefor supporting a replacement prosthesis. Furthermore, the presence ofcontaminating microorganisms in surrounding tissues makesrecontamination of the replacement prosthesis easily possible. Theeffects of repeated contamination and infection of structuralprosthetics is significant: major reconstructive surgery may be requiredto rehabilitate the area in the absence of the prosthesis, potentiallyincluding free bone transfers or joint fusions. Furthermore, there is noguarantee that these secondary reconstructive efforts will not meet withinfectious complications as well. Major disability, with possibleextremity amputation, is the outcome from contamination and infection ofa structural prosthesis.

Certain implantable devices are intended to restore or enhance bodycontours for cosmetic or reconstructive applications. A well-knownexample of such a device is the breast implant, a gel or fluidcontaining sac made of a silicone elastomer. Other polymeric implantsexist that are intended for permanent cosmetic or reconstructive uses.Solid silicone blocks or sheets can be inserted into contour defects.Other naturally occurring or synthetic biomaterials are available forsimilar applications. Craniofacial surgical reconstruction can involveimplantable devices for restoring severely deformed facial contours inaddition to the techniques used for restructuring natural bony contours.These devices, and other related devices well-known in the field, aresuitable for coating with or impregnation with sulfate ester AF agentsto reduce their risk of contamination, infection and subsequent removal.

Tissue expanders are sacs made of silicone elastomers adapted forgradual filling with a saline solution, whereby the filling processstretches the overlying tissues to generate an increased area of tissuethat can be used for other reconstructive applications. Tissue expanderscan be used, for example, to expand chest wall skin and muscle aftermastectomy as a step towards breast reconstruction. Tissue expanders canalso be used in reconstructing areas of significant skin loss in burnvictims. A tissue expander is usually intended for temporary use: oncethe overlying tissues are adequately expanded, they are stretched tocover their intended defect. If a tissue expander is removed before theexpanded tissues are transposed, though, all the expansion gained overtime is lost and the tissues return nearly to their pre-expansion state.The most common reason for premature tissue expander removal isinfection. These devices are subjected to repeated inflations of salinesolution, introduced percutaneously into remote filling devices thatcommunicate with the expander itself. Bacterial contamination of thedevice is thought to occur usually from the percutaneous inflationprocess. Once contamination is established and a biofilm forms, localinfection is likely. Expander removal, with the annulment of thereconstructive effort, is needed to control the infection. A delay of anumber of months is usually recommended before a new tissue expander canbe inserted in the affected area. The silicone elastomer used for thesedevices is especially suitable for integrating with sulfate ester AFagents. Use of these agents in the manufacture of these articles mayreduce the incidence of bacterial contamination, biofilm development andsubsequent local infection.

Insertable devices include those objects made from synthetic materialsapplied to the body or partially inserted into the body through anatural or an artificial site of entry. Examples of articles applied tothe body include contact lenses and stoma appliances. An artificiallarynx is understood to be an insertable device in that it exists in theairway, partially exposed to the environment and partially affixed tothe surrounding tissues. An endotracheal or tracheal tube, a gastrostomytube or a catheter are examples of insertable devices partially existingwithin the body and partially exposed to the external environment. Theendotracheal tube is passed through an existing natural orifice. Thetracheal tube is passed through an artificially created orifice. Underany of these circumstances, the formation of biofilm on the devicepermits the ingress of microorganisms along the device from a moreexternal anatomic area to a more internal anatomic area. The ascent ofmicroorganisms to the more internal anatomic area commonly causes localand systemic infections.

As an example, biofilm formation on soft contact lenses is understood tobe a risk factor for contact-lens associated corneal infection. The eyeitself is vulnerable to infections due to biofilm production.Incorporating an antifouling agent in the contact lens itself and in thecontact lens case can reduce the formation of biofilms, thereby reducingrisk of infection. Sulfate ester AF agents can also be incorporated inophthalmic preparations that are periodically instilled in the eye.

As another example, biofilms are understood to be responsible forinfections originating in tympanostomy tubes and in artificial larynxes.Biofilms further reside in tracheostomy tubes and in endotracheal tubes,permitting the incursion of pathogenic bacteria into the relativelysterile distal airways of the lung. These devices are adaptable to theincorporation or the topical application of sulfate ester AF agents toreduce biofilm formation and subsequent infectious complications.

As another example, a wide range of vascular catheters are fabricatedfor vascular access. Temporary intravenous catheters are placeddistally, while central venous catheters are placed in the more proximallarge veins. Catheter systems can include those installed percutaneouslywhose hubs are external to the body, and those whose access ports areburied beneath the skin. Examples of long-term central venous cathetersinclude Hickman catheters and Port-a-caths. Catheters permit theinfusion of fluids, nutrients and medications; they further can permitthe withdrawal of blood for diagnostic studies or the transfusion ofblood or blood products. They are prone to biofilm formation,increasingly so as they reside longer within a particular vein. Biofilmformation in a vascular access device can lead to the development of ablood-borne infection as planktonic organisms disseminate from thebiofilm into the surrounding bloodstream. Further, biofilm formation cancontribute to the occlusion of the device itself, rendering itnon-functional. If the catheter is infected, or if the obstructionwithin it cannot be cleared, the catheter must be removed. Commonly,patients with these devices are afflicted with serious medicalconditions. These patients are thus poorly able to tolerate the removaland replacement of the device. Furthermore, there are only a limitednumber of vascular access sites. A patient with repeated catheterplacements can run out of locations where a new catheter can be easilyand safely placed. Incorporation of sulfate ester AF agents withincatheter materials or application of these agents to catheter materialscan reduce fouling and biofilm formation, thereby contributing toprolonged patency of the devices and minimizing the risk of infectiouscomplications.

As another example, a biliary drainage tube is used to drain bile fromthe biliary tree to the body's exterior if the normal biliary system isblocked or is recovering from a surgical manipulation. Drainage tubescan be made of plastics or other polymers. A biliary stent, commonlyfabricated of a plastic material, can be inserted within a channel ofthe biliary tree to keep the duct open so that bile can pass through it.Biliary sludge which forms as a result of bacterial adherence andbiofilm formation in the biliary system is a recognized cause ofblockage of biliary stents. Pancreatic stents, placed to hold thepancreatic ducts open or to drain a pseudocyst of the pancreas, can alsobecome blocked with sludge. Biofilms are furthermore implicated in theascent of infections into the biliary tree along a biliary drainagetube. Ascending infections in the biliary tree can result in thedangerous infectious condition called cholangitis. Incorporation ofcompounds of the invention in the materials used to form biliarydrainage tubes and biliary stents can reduce the formation of biofilms,thereby decreasing risk of occlusions and infections.

As another example, a peritoneal dialysis catheter is used to removebodily wastes in patients with renal failure by using fluids instilledinto and then removed from the peritoneal cavity. This form of dialysisis an alternative to hemodialysis for certain renal failure patients.Biofilm formation on the surfaces of the peritoneal dialysis cathetercan contribute to blockage and infection. An infection entering theperitoneal cavity is termed a peritonitis, an especially dangerous typeof infection. Peritoneal dialysis catheters, generally made of polymericmaterials like polyethylene, can be coated with or impregnated withsulfate ester AF agents to reduce the formation of biofilms.

As yet another example, a wide range of urological catheters exist toprovide drainage of the urinary system. These catheters can either enterthe natural orifice of the urethra to drain the bladder, or they can beadapted for penetration of the urinary system through an iatrogenicallycreated insertion site. Nephrostomy tubes and suprapubic tubes representexamples of the latter. Catheters can be positioned in the ureters on asemipermanent basis to hold the ureter open; such a catheter is called aureteral stent. Urological catheters can be made from a variety ofpolymeric products. Latex and rubber tubes have been used, as havesilicones. All catheters are susceptible to biofilm formation. Thisleads to the problem of ascending urinary tract infections, where thebiofilm can spread proximally, carrying pathogenic organisms, or wherethe sessile organisms resident in the biofilm can propagate planktonicorganisms that are capable of tissue and bloodstream invasion. Organismsin the urinary tract are commonly gram-negative bacteria capable ofproducing life-threatening bloodstream infections if they spreadsystemically. Infections wherein these organisms are restricted to theurinary tract can nonetheless be dangerous, accompanied by pain and highfever. Urinary tract infections can lead to kidney infections, calledpyelonephritis, that can jeopardize the function of the kidney.Incorporating sulfate ester AF agents can inhibit biofilm formation andmay reduce the likelihood of these infectious complications.

A further complication encountered in urological catheters isencrustation, a process by which inorganic compounds comprising calcium,magnesium and phosphorous are deposited within the catheter lumen,thereby blocking it. These inorganic compounds are understood to arisefrom the actions of certain bacteria resident in biofilms on cathetersurfaces. Reducing biofilm formation by the action of sulfate ester AFagents may contribute to reducing encrustation and subsequent blockageof urological catheters.

Other catheter-like devices exist that can be treated with AF agents.For example, surgical drains, chest tubes, hemovacs and the like can beadvantageously treated with materials to impair biofilm formation. Otherexamples of such devices will be familiar to ordinary practitioners inthese arts.

Materials applied to the body can advantageously employ the AF compoundsdisclosed herein. Dressing materials can themselves incorporate the AFcompounds, as in a film or sheet to be applied directly to a skinsurface. Additionally, AF compounds of the instant invention can beincorporated in the glue or adhesive used to stick the dressingmaterials or appliance to the skin. Stoma adhesive or medical-grade gluemay, for example, be formulated to include an AF agent appropriate tothe particular medical setting. Stoma adhesive is used to adhere stomabags and similar appliances to the skin without traumatizing the skinexcessively. The presence of infectious organisms in these appliancesand on the surrounding skin makes these devices particularly appropriatefor coating with AF agents, or for incorporating AF agents therein.Other affixation devices can be similarly treated. Bandages, adhesivetapes and clear plastic adherent sheets are further examples where theincorporation of an AF agent in the glue or other adhesive used to affixthe object, or incorporation of an AF agent as a component of the objectitself, may be beneficial in reducing skin irritation and infection.

These above examples are offered to illustrate the multiplicity ofapplications of compounds of the invention in medical devices. Otherexamples will be readily envisioned by skilled artisans in these fields.The scope of the present invention is intended to encompass all thosesurfaces where the presence of fouling has adverse health-relatedconsequences. The examples given above represent embodiments where thetechnologies of the present invention are understood to be applicable.Other embodiments will be apparent to practitioners of these and relatedarts. Embodiments of the present invention can be compatible forcombination with currently employed antiseptic regimens to enhance theirantimicrobial efficacy or cost-effective use. Selection of anappropriate vehicle for bearing a compound of the invention will bedetermined by the characteristics of the particular medical use. Otherexamples of applications in medical environments to promote antisepsiswill be readily envisioned by those of ordinary skill in the relevantarts.

The present invention is further illustrated by the following examples,which should not be construed as limiting in any way. The contents ofall cited references including literature references, issued patents andpublished patent applications as cited throughout this patentapplication are hereby expressly incorporated by reference.

Examples Example 1

Inhibition of Surface Attachment of Marine Bacteria by Alkyl Sulfates

Octyl sulfate is an alkyl sulfate surfactant with extensive industrialapplications, and is manufactured by several large chemical companies.To investigate potential AF properties of the sulfate ester octylsulfate, it was incorporated into an inert coating material that wasthen coated onto a surface to be exposed to conditions that support theformation of marine algal biofilms.

Materials and Methods

A 30% (w/v) solution of octyl sulfate in water (Stepan Chemical Co.) wasevaporated to dryness under a stream of room temperature air, to recoverpure octyl sulfate (FIG. 1). The dry octyl sulfate was incorporated intoRTV-11 silicone polymer at a loading of 25% (wt/wt) (RTV-11 silicone,catalyst and primer obtained from General Electric). The mixture wasapplied to three glass slides previously primed with silicone primer,and allowed to cure to dryness. Three primed glass slides coated withpure RTV-11 served as agent-free controls. After complete drying, theabsorption properties of each slide were measured using a ShimadzuUV-2101 spectrophotometer fitted with an integrating sphere. Slides werethen placed in a tank of running raw seawater and allowed to incubateoutdoors in natural sunlight for 26 days. Water temperature wasnominally 15 C. Spectrophotometric determination of biofilm accumulationwas measured on each slide periodically. Relative algal biomass wascalculated as the ratio of absorption at 680 nm, contributed bychlorophyll a, to that at 750 nm, a wavelength not absorbed bychlorophyll, to correct for differences in turbidity and scatteringproperties of the different slides.

Results

As shown in FIG. 1, octyl sulfate incorporated into RTV-11 silicone, andthen coated onto glass slides, significantly inhibited the formation ofnatural marine algal biofilms in natural seawater. After 26 days ofincubation in running seawater, algal biofilm development on the octylsulfate containing coatings was five fold less than that of controlslacking octyl sulfate, indicating that octyl sulfate possesses strong AFactivity.

Studies were performed to evaluate the ability of the sulfate estermolecules octyl sulfate and methyl sulfate, to inhibit adhesion of themarine bacteriums Oceanosprillum and Alteromonas atlantica to glasssurfaces.

Materials and Methods

Oceanosprillum adhesion test Each test consisted of a control set (withno sulfate esters) and sample sets containing the test molecules. Thefirst test group consisted of a control sample set, a zosteric acid (5mM) sample set, and an octyl sulfate (5 mM) sample set. The second testgroup consisted of a control sample set, a zosteric acid (5 mM) sampleset, and a methyl sulfate (5 mM) sample set. Sample sets consisted offive 50 mL sterile centrifuge tubes, with each tube containing a glassmicroscope slide, 50 ml of artificial seawater (ASTM—American Societyfor testing and materials) with the dissolved sulfate ester, inoculatedwith an Oceanosprillum culture at 1×10⁶ cells/mL. Sample sets wereincubated at 23 C, with shaking so that the surface of the slides werehorizontal. Over a 6-hour period, individual tubes were taken from thesample sets and tested for microbial adhesion.

Alteromonas atlantica adhesion tests. The tests consisted of a controlsample set, a zosteric acid (5 mM) sample set, an octyl sulfate (5 mM)sample set, and a methyl sulfate (5 mM) set. A sample set consisted ofsix 60 mL sterile centrifuge tubes. Each tube contained a glassmicroscope slide and 50 mL of modified ASTM seawater (American Societyfor Testing and Materials (1986) D1141-86, ASTM, Philadelphia, Pa.) withdissolved agent, inoculated with Alteromonas atlantica culture to aninitial cell density of 1×10⁶ cells/mL. The modified seawater consistedof normal ASTM seawater ingredients, however the carbon source glycerol,was only 1000th the normal strength, 0.1 L/L instead of 100 L/L, and wasvoid of an amino acid source (casamino acids), in order to allow enoughcarbon for attachment, but not for significant cell growth.

Determination of bacterial adhesion. Samples were removed from theshaker and 1 mL of 50X acridine orange stain (0.5 g/L acridine powder inwater) was added to the tube. The stain was allowed to react for 4minutes. The slides were then removed and fitted with a long cover slipand immediately counted with an epifluorescent microscope fitted with a100X (oil) objective lens on the under side of the slide. The size ofthe counting field was 10 X 10 μm. A total of 20 counts per slide wereperformed and averaged to yield the number of cells per μm², which wasin turn converted to cells per mm². Error was assigned at 10% which isthe standard accepted error for direct counting of bacterial cells.

Results

As shown in FIG. 2, the presence of octyl sulfate or methyl sulfate inthe medium significantly reduced bacterial adhesion to the glass slideswhen compared to controls in which no sulfate ester molecule waspresent. Methyl sulfate inhibited Oceanosprillum adhesion to an extentsimilar to the proven AF agent zosteric acid, with each compoundpromoting roughly a two fold reduction in bacterial attachment, relativeto control. As shown in FIG. 3, octyl sulfate inhibited Oceanosprillumadhesion to an even greater extent than zosteric acid.

As shown in FIG. 4, the presence of dissolved zosteric acid, octylsulfate, or methyl sulfate produced a significant reduction in themarine bacterium, Alteromonas atlantica adhesion relative the controls.The presence of methyl sulfate had the most dramatic effect uponadhesion, with adhesion remaining constant after 120 minutes at 150,000cells/mm², while controls had greater than 700,000 cells/mm². Octylsulfate also inhibited adhesion, demonstrating a slightly higherinhibitory activity than zosteric acid.

Example 2

Inhibition of Fungal Surface Attachment and Mycelial Development

To determine the effectiveness of sulfate esters at inhibiting fungalbiofouling, the ability of zosteric acid to inhibit attachment of thefungus Aureobasidium pullulans to surfaces was examined.

Materials and Methods

Aureobasidium pullulans (ATCC 34261) was grown on potato-dextrose agarand harvested according to ASTM G-21-90 protocols (American Society forTesting and Materials (1986) D1141-86, ASTM, Philadelphia, Pa.). Theresulting spore suspension was used to inoculate liquid culture tubescontaining 35 mL of growth medium (nutrient salts with 5 mM sucrose) and15 mM zosteric acid. Zosteric acid-free medium was prepared as acontrol. A sterile microscope slide was added to each tube, the tubeswere sealed and placed on a rotary shaker table at room temperature. Onetube was harvested each day by removing the slide and counting thenumber of attached spores by direct microscopic counts, as describedabove.

Results

Fungal spores were observed to grow in both the presence and absence ofzosteric acid, as indicated by the clouding of all tubes after Day 1.However, as shown in FIG. 5, the presence of zosteric acid prevented theattachment of the fungus to the glass slides. After 5 days incubationwith A. pullalans, less than 20 germinated fungal colonies/mm² wereobserved on slides incubated in the additional presence of zostericacid, compared to more than 600 germinated fungal colonies/mm² oncontrol slides. Furthermore, fungal colonies in the media of zostericacid free cultures were composed of multi-cellular (>20 cells)filaments, indicative of mycelial growth. In contrast, colonies in thezosteric acid treated cultures were generally small and round,exhibiting no evidence of filamentous growth or mycelial development.

Example 3

Sulfate Esters Bind Cell Surfaces of Biofouling Organisms

To investigate the mechanism behind the AF activity of sulfate esters,polyclonal antibodies specific for the sulfate ester zosteric acid weregenerated (BAbCo, Berkeley, Calif.). Preliminary testing of theseantibodies for cross reactivity towards related compounds lacking thesulfate ester group (cinnamic acid, ferulic acid, coumaric acid) showedno cross reactivity, suggesting that the specific domain recognized bythe antibodies probably includes the sulfate ester group. Theseantibodies were then used to investigate whether the sulfate ester AFagent zosteric acid directly binds fouling organisms.

The marine bacterium Shewanella putrefaciens were grown in culturescontaining zosteric acid and were subsequently examined for boundzosteric acid using immuno-gold staining with the antibody describedabove. Electron microscopic examination of immunoprobed S. putrefaciensdetected zosteric acid molecules bound to the surface of the bacteria.Furthermore, zosteric acid was observed to be present at high incidenceat the sites of cell adhesion. In contrast to these agglutination sites,the majority of the cell surfaces as well as the continuous boundariesbetween daughter cells in dividing chains, showed no evidence of boundzosteric acid, as indicated by a lack of immuno-gold staining. Theseresults indicate that sulfate esters bind to the surfaces of bacterialcells and suggest a possible relationship between sulfate ester bindingsites and the sites of bacterial agglutination.

Example 4

Zosteric Acid Promotes Bacterial Agglutination

To further investigate the role of sulfate esters in agglutination, theability of sulfate esters to facilitate the agglutination of bacterialcells was investigated. Log-phase cultures grown in the presence ofzosteric acid were monitored spectrophotometrically (OD₆₀₀) for growth,and for agglutination in the presence of increasing amounts of zostericacid.

Materials and Methods

Cell Surface Binding Assays. The marine bacterium Shewanellaputrefaciens was grown in marine broth in the presence of 16mM zostericacid. Dense log phase cells were harvest after 5 hours growth, andpreserved in 0.5 X Karnofsky's fixative (2% formaldehyde, 2.5%gluturaldehyde, 0.05 M sodium cacodylate, 0.25 M sucrose, pH 7.4) for 2hours, and then transferred to a cacodylate buffer (0.05 M sodiumcacodylate, pH 7.4) for storage. Cells were prepared for electronmicroscopic examination using immuno-gold staining techniques (Harlow,E. and Laine, D., Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, 359-421; Roth et al., J. Histochem. Cytochem. 26: 1074-1081(1978)). The primary antibody used in this study was an anti-zostericacid polyclonal antibody (BAbCo, Richmond, Calif.).

Bacterial agglutination assays. Log-phase cultures of Shewanellaputrefaciens were grown in complete seawater medium containing zostericacid at a range of concentrations up to 20 mM. Cultures were counted forviable colony forming units at eight hours.

Results

Although zosteric acid concentrations up to 16 mM did not inhibit thegrowth of S. putrefaciens in liquid culture, the presence of zostericacid caused significant agglutination of S. putrefaciens in aconcentration dependent manner. The agglutination observed was visibleto the naked eye, and was more quantitatively detected as a decrease inoptical density absorbance in cultures containing zosteric acid (FIG.6). Counts of viable colony forming units at eight hours revealed nodifference in cell density among the different cultures, thus theobserved differences in absorption resulted from differences inbacterial agglutination, not differences in growth (cell division) ratesamong the cultures. Thus, zosteric acid promoted cell agglutination, butdid not affect cell growth.

Example 5

Zosteric Acid Binds Heparin-sensitive Sites

To investigate the ability of sulfate esters to mediate the interactionbetween biological surfaces involved in erythrocyte agglutination andblood clotting, erythrocyte agglutination assays and clot formationassays were performed using the sulfate ester zosteric acid.

Materials and Methods

Red Blood Cell agglutination assays. Washed equine erythrocytessuspended in 1 mg/mL sodium citrate were placed in microtiter platesdesigned with wells containing hemispherical bottoms. Negative controls(no zosteric acid) were diluted in isotonic saline solution. Zostericacid treated cells were diluted with saline containing zosteric acid ateight concentrations ranging from 0.005 to 5.0 mg/mL. Positive controlswere exposed to the same range of high molecular weight heparin sulfateconcentrations.

Clotting assays. Clotting time assays were performed using commercialkits (Sigma Chemical Co.) for prothrombin clotting time. Serum washarvested from 30 mL of whole human blood obtained by venous punctureusing centrifugation to remove blood cells. Zosteric acid and high MWheparin were added to separate aliquots of the serum, producingconcentrations from 0 to 5.0 mg/mL. Clotting times were determined foreach concentration in duplicate according to the protocols provided withthe kit.

Results

In agglutination studies, equine erythrocytes were significantlyagglutinated by zosteric acid at concentrations as low as 0.175 mg/mL.In contrast, the presence of high molecular weight heparin producedvisible agglutination only at concentrations greater than 0.75 mg/mL.This result indicates that monomeric zosteric acid is eight times morereactive with cell surface glycoproteins and polysaccharides involved incell agglutination, than high molecular weight heparin.

Zosteric acid was also effective at preventing clot formation, asmeasured by the prothrombin clotting time assays (FIG. 7), although thisactivity was considerably less than that observed for heparin. Heparinwas effective at preventing clot formation at concentrations well below0.1 mg/ml, while zosteric acid was effective only at concentrationsexceeding 10 mg/ml. The effectiveness of heparin-like anticoagulants isstrongly linked to size, with high molecular weight molecules being moreeffective. Thus, it is not surprising that the low molecular weightzosteric acid was considerably less effective than high molecular weightheparin in mediating clot formation. A derivative of zosteric acid oranother sulfate ester that is higher in molecular weight may prove moreeffective. Nonetheless, these results indicate that zosteric acidinteracts with cell surface glycoproteins and/or polysaccharides in amanner similar to that of heparin.

Example 6

Zosteric Acid Blocks Fertilization

The data above suggests that sulfate esters interact with sulfateester-binding receptors in a variety of systems ranging from bacteria tomammalian erythrocytes. The fusion of sperm and egg cells ininvertebrate and mammalian systems also appears to be mediated byorgano-sulfate molecules such as the polysaccharides fucose sulfate andheparin. In light of this, the following experiments were initiated toidentify potential AF properties of sulfate esters in fertilization.

A simple sea urchin assay was used to detect and quantitate the abilityof sulfate esters to block sperm-egg fusion. Sea urchin sperm was addedto freshly collected eggs in the presence and absence of increasingamounts of the sulfate ester zosteric acid, and the eggs weresubsequently scored for successful fertilization.

Materials and Methods

Fertilization assays. Healthy sea urchins were induced to spawn byinjection with 0.5 M KCL solution. Freshly collected eggs were gentlywashed and resuspended in filtered sea water (FSW, pH 8.2) andaliquotted into separate tubes for fertilization assays. Zosteric acidwas added to each tube from a concentrated stock dissolved in FSW (pH8.2), along with additional FSW to ensure a constant volume in eachtube. Equal amounts of sperm were added to each tube and percentfertilization was determined by direct microscopic counting. Eggs withelevated fertilization membranes were scored as fertilized. Assays wereperformed at sperm-limiting concentrations that allowed 95-99%fertilization in the absence of zosteric acid.

Sea urchin egg agglutination assays. Agglutination of unfertilized eggsby bindin was evaluated at the range of zosteric acid concentrationsindicated in Table 1. Freshly spawned eggs were suspended in acidicseawater (pH 5) for 5 minutes to remove the outer jelly coat, and thenwashed 5 times in normal FSW (pH 8.2). Eggs were then transferred intoplastic petri dished containing a range of zosteric acid concentrationsand incubated for 15 minutes. Purified bindin (D. Epel, StanfordUniversity) was added to the eggs at concentrations ranging from 1.2 to12 μg/mL. The mixtures were gently agitated on a rotary shaker for 5minutes and visually examined for agglutination. Bovine serum albumen(BSA) was used in separate assays to control for nonspecificagglutination of the dejellied eggs.

Dot blot assays. Serial dilutions of purified bindin, a covalentlyconjugated zosteric acid-BSA molecule, and an unconjugated mixture offree zosteric acid and BSA were pipetted onto a nitrocellulose membraneand allowed to air-dry. Standard immuno-blotting procedures were thenemployed to determine the reactivity between the blotted substrates anda polyclonal anti-zosteric acid antibody. The membrane was blocked inblotto (1% nonfat dry milk in phosphate buffered saline (PBS)) for 1hour prior to probing. Probing was done in blotto for 1 hour. Primaryantibody was anti-zosteric acid antibody, used at a 1:1000 dilution.Secondary antibody was alkaline phosphatase conjugated goat anti-rabbit(Southern Biotechnology Association, Inc.) and was used at a dilution of1:1000. Rinses between probing were performed in triplicate inPBS-tween. The blot was developed in color reaction buffer (100 mM Tris,pH 9.5, 100 mM NaCl, 5 mM MgCl₂, 50 mg/niL Nitroblue Tetrazolium(Sigma), 50 ml/mL 5-bromo-4-chloro-3-indolyl phosphate (BCIP, Sigma))for 20 minutes. Membranes were then transferred to stop buffer (10 mMTris, pH 6.0, 5 mM EDTA) for 1 hour, and then transferred to freshwater,left overnight, and then dried.

Results

As shown in FIG. 8 a, zosteric acid had a dose-dependent effect on seaurchin egg fertilization. Concentrations higher than 0.5 mg/mL (1.5 mM)completely blocked fertilization. Re-exposure of unfertilized eggs fromthe highest zosteric acid treatment, to fresh sperm, after washing insea water, resulted in the fertilization of all eggs. This resultdemonstrates that the zosteric acid inhibition is reversible. Thepresence of zosteric acid had no detected effect on sperm viability ormotility. Sperm exposed to zosteric acid were observed to swim activelythrough the jelly layer surrounding the egg without adhering to the eggsurface or elevating the egg fertilization membrane, further supportingthe conclusion that the antifouling effect of zosteric acid was mediatedthrough inhibition of sperm-egg attachment.

The effectiveness of zosteric acid (1 mg/mL) at fertilization inhibitionwas compared to equal mass concentrations of coumaric acid (anunsulfated zosteric acid precursor) and high MW heparin. The presence ofcoumaric acid had no effect on egg fertilization, while the presence ofheparin reduced fertilization by approximately 50%. Zosteric acid was atleast twice as effective as heparin at inhibiting fertilization,reducing fertilization to 21% at this concentration (FIG. 8 b).

The ability of zosteric acid to compete for the binding of sulfatereceptor sites on the egg surface was investigated in egg agglutinationassays. These experiments tested the ability of zosteric acid tointerfere with the binding of the bindin molecule to unfertilized seaurchin eggs. Bindin added to unfertilized eggs causes them toagglutinate by cross linking sulfate receptors that are present on thesurface of the eggs. Addition of zosteric acid inhibited thisagglutination in a dose dependent manner (Table 1), suggesting acompetitive interaction of bindin and zosteric acid for the sulfatereceptor sites on the egg surface. [ZA] mg mL⁻¹ Agglutination 3 No 1.5No 0.75 Yes 0.30 Yes 0.15 Yes 0.075 Yes 0.03 Yes 0.015 YesTable 1. The effect of zosteric acid on the agglutination of sea urchineggs by purified bindin.

Antibodies specific for zosteric acid (described above) exhibited strongcross reactivity with the bindin molecule in dot-blot assays, but notwith other proteins, such as bovine serum albumin. This antibody crossreactivity indicates that zosteric acid and bindin share significantstructural similarity at the site of antibody recognition, believed tobe the sulfate moiety. Such structural similarities in the sulfatemoieties between bindin and zosteric acid would explain why zostericacid is an effective inhibitor of sea urchin fertilization.

Example 7

4 t-Pentyl Phenyl Chlorosulfate (4-PPCS) Blocks Fertilization

The effect of 4-PPCS on inhibiting sea urchin fertilization wasperformed substantially as described for zosteric acid in Example 6. Ascan be seen in FIG. 7, 4-PPCS was essentially 100% effective in blockingsea urchin fertilization in the range of 1-10 mM, precisely the samerange as zosteric acid was effective. As also can be seen in FIG. 7,when more sperm were added to the medium, the effect of the PPCSinhibition could be washed out be exceeding the binding capacity of the4-PPCS in solution. In contrast to most biocidal agents, contact with4-PPCS resulted in no adverse impacts on sperm motility.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1-86. (canceled)
 87. A method of preventing fouling on the surface of amedical device comprising contacting the surface with an effectiveamount of anti-fouling compound represented by general structure 1:

wherein X represents —OH, F, Cl, or Br; Y represents O, S, or NR; Zrepresents optionally substituted alkyl, aryl, heteroaryl, or aralkyl; Rrepresents independently for each occurrence hydrogen, alkyl, or aryl.88. The method of claim 87, wherein X is OH or Cl.
 89. The method ofclaim 87, wherein Y is O.
 90. The method of claim 87, wherein Z isalkyl.
 91. The method of claim 87, wherein Z is aryl.
 92. The method ofclaim 87, wherein X is OH or Cl, and Y is O.
 93. The method of claim 87,wherein X is OH or Cl, Y is O, and Z is alkyl.
 94. The method of claim87, wherein X is OH or Cl, Y is O, and Z is aryl.
 95. The method ofclaim 87, wherein Z is methyl, n-octyl, phenyl,4-(2-methylpropyl)phenyl, 4-(1,1-dimethylethyl)phenyl,4-(1,1-dimethylpropyl)phenyl, 4-pentylphenyl,4-(1-methyl-1-phenylethyl)phenyl, or 4-(1-methylheptyl)phenyl.
 96. Themethod of claim 87, wherein X is OH, Y is O, and Z is methyl.
 97. Themethod of claim 87, wherein X is OH, Y is O, and Z is n-octyl.
 98. Themethod of claim 87, wherein X is OH, Y is O, and Z is phenyl.
 99. Themethod of claim 87, wherein X is Cl, Y is O, and Z is 4-t-butylphenyl.100. The method of claim 87, wherein X is Cl, Y is O, and Z is4-t-pentylphenyl.
 101. The method of claim 87, wherein the anti-foulingcompound is dissolved or dispersed in a medium.
 102. The method of claim103, wherein the medium comprises phenolic resin, silicone polymer,epoxy resin, polyamide resin, vinyl resin, elastomer, acrylate polymer,silicone resin, polyester, chlorinated rubber, polyurethane, latex, orfluoropolymer.
 103. The method of claim 103, wherein the medium is anaqueous medium.
 104. The method of claim 87, wherein contacting thesurface comprises spraying, wetting, immersing, dipping, or painting thesurface.
 105. The method of claim 87, wherein the medical device is ascalpel, needle, scissors, catheter, orthopedic pins, artificial heart,artificial kidney, implant, plate, prostheses, glove, apron, faceshield,counter top, or water tube.